Compositions and methods of using tyrosine kinase inhibitors

ABSTRACT

The present invention provides compositions and methods of inhibiting tyrosine phosphorylation. In one aspect, a composition comprising a low-dosage tyrosine kinase inhibitor, where the low-dosage tyrosine kinase inhibitor decreases tyrosine phosphorylation, is provided. In another aspect, a method for treating cardiovascular disease or condition associated with a RASopathy having aberrant protein tyrosine phosphorylation is described. Methods for treating congenital heart disease associated with Noonan or Noonan syndrome with multiple lentigines and decreasing aberrant levels of Protein Zero-Related (PZR) tyrosyl phosphorylation are also described.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 62/250,052, filed Nov. 3, 2015, and U.S. Provisional ApplicationSer. No. 62/107,553, filed Jan. 26, 2015, the contents of which areincorporated by reference herein in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under GM099801 awardedby the National Institute of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

Cardiovasular disease is the leading cause of death for both men andwomen worldwide despite significant advances. According to a report bythe World Health Organization (WHO), it is estimated that 23.6 millionpeople will die from cardiovascular diseases annually by 2030.

The RAS-MAPK pathway is critical for human growth and development.Abnormalities at different steps of this signaling cascade result inneuro-cardio-facial-cutaneous syndromes, or the RASopathies, a group ofdisorders with overlapping yet distinct phenotypes. RASopathy patientshave variable degrees of intellectual disability, poor growth, relativemacrocephaly, ectodermal abnormalities, dysmorphic features, andincreased risk for certain malignancies. Significant locus heterogeneityexists for many of the RASopathies.

Congenital heart disease (CHD) is the most common defect found innewborns, occurring in about 1% of live births. Over 1 million people inthe United States have some form of CHD, most of whom require continualmonitoring and treatment to prevent deterioration of cardiac function.AVCD includes different anomalies of atrioventricular valves and atrialand ventricular septa. In the complete form, a single commonatrioventricular valve and an atrial septal defect (ostium primum)confluent with a posterior ventricular septal defect in the inletportion of the ventricular septum are found. In the partial form, thereare two separate right and left atrioventricular valves with a cleftedmitral valve, an atrial septal defect (ostium primum), and noventricular septal communication. Cleft mitral valve is considered theless severe form of AVCD. AVCD is also the most common CHD found inchildren with Down syndrome and one of the structural heart defects mostfrequently associated with extracardiac anomalies in the setting ofchromosomal and mendelian disorders. Distinct anatomic features arefound in AVCD associated with NS. In fact, in general this defect is ofthe partial type, eventually associated with subaortic stenosis, due toaccessory fibrous tissue and/or anomalous insertion of the mitral valvewith anomalous papillary muscle of the left ventricle.

Congenital heart disease (CHD) occurs in approximately 60-86% ofpatients affected by a RASopathy, a group of disorders withabnormalities in the RAS-MAPK pathway. Pulmonary valve stenosis (PVS)and hypertrophic cardiomyopathy are the most common defects displaying adistinct association with the RASopathies. The spectrum of CHDs inNoonan syndrome with multiple lentigines (NSML) is wider, and the familyof atrioventricular canal defects (AVCD) is the third most common heartdefect.

Most patients with cardiovascular disease and RASopathy-associatedcongenital heart disease need treatment for many years. In particular,RASopathy-associated congenital heart disease are usually associatedwith low mortality rates. Therefore a need exists to treatcardiovascular disease in patients with low risk therapies havingmaximal effect on heart disease.

SUMMARY OF THE INVENTION

As described below, the present invention includes compositions andmethods to aberrant inhibit protein tyrosine phosphorylation, such asphosphorylation of Src family tyrosine kinases and their substrates.

In one aspect, the invention includes a method of treating acardiovascular disease or condition having aberrant protein tyrosinephosphorylation in a subject, comprising administering a low-dosage of atyrosine kinase inhibitor to a subject in need thereof, wherein thetyrosine kinase inhibitor decreases aberrant levels of tyrosinephosphorylation and improves at least one cardiac function in thesubject.

In another aspect, the invention includes a method of treatingcongenital heart disease comprising administering a low-dosage of atyrosine kinase inhibitor to a subject in need thereof, wherein thetyrosine kinase inhibitor decreases aberrant levels of tyrosinephosphorylation and improves at least one cardiac function in thesubject.

In yet another aspect, the invention includes a method of treating acardiovascular disease or condition associated with a RASopathy havingaberrant protein tyrosine phosphorylation comprising administering alow-dosage of a tyrosine kinase inhibitor to a subject in need thereof,wherein the tyrosine kinase inhibitor decreases aberrant levels oftyrosine phosphorylation and improves at least one cardiac function inthe subject.

In still another aspect, the invention includes a composition comprisinga low-dosage tyrosine kinase inhibitor, wherein the low-dosage tyrosinekinase inhibitor is capable of decreasing tyrosine phosphorylation andimproving at least one cardiac function in a subject in need thereof.

In another aspect, the invention includes a pharmaceutical compositioncomprising the composition as described herein and a pharmaceuticallyacceptable carrier.

In yet another aspect, the invention includes use of the composition asdescribed herein in the manufacture of a medicament for the treatment ofcardiovascular disease or condition in a subject.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the congenital heart disease is associatedwith a RASopathy, such as a RASopathy selected from the group consistingof Neurofibromatosis Type 1, Noonan syndrome, Noonan syndrome withmultiple lentigines (Leopard syndrome), capillarymalformation-arteriovenous malformation syndrome, Costello syndrome,cardio-facio-cutaneous syndrome, and Legius syndrome. In one embodiment,the cardiovascular disease or condition is congenital heart disease.

In another embodiment, the low-dosage is in the range of about 175 foldto about 250 fold lower than a chemotherapeutic dosage of the tyrosinekinase inhibitor.

In another embodiment, the cardiac function is selected from the groupconsisting of myofibrilar organization, cardiomyocyte contractility,SERCA2A expression, and cardiac fibrosis.

In another embodiment, the tyrosine kinase inhibitor is selected fromthe group consisting of afatinib, axitinib, bosutinib, cabozantinib,cediranib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib,everolimus, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib,lestaurtinib, nilotinib, nintedanib, palbociclib, pazopanib, ponatinib,regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, sunitinib,temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib. Inyet another embodiment, the tyrosine kinase inhibitor is a Src familytyrosine kinase inhibitor, such as a Src family tyrosine kinaseinhibitor selected from the group consisting A419259, AP23451, AP23464,AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285,PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003,TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228, altenusin,bosutinib, damnacanthal, dasatinib, herbimycin A, indirubin, neratinib,lavendustin A, pelitinib, piceatannol, saracatinib, SrcI1, and analogsthereof.

In another embodiment, the subject is a pediatric patient, such as apediatric subject less than 12 years of age. In yet another embodiment,the subject is greater than 18 years of age.

In another embodiment, the aberrant levels of tyrosine phosphorylationcomprise aberrant levels of tyrosine phosphorylated Protein Zero-Related(PZR). In such an embodiment, the low-dosage tyrosine kinase inhibitordecreases PZR tyrosine phosphorylation. In yet another embodiment, thelow-dosage tyrosine kinase inhibitor provides an anti-fibrotic effect incardiac tissue to the subject. In still another embodiment, thelow-dosage tyrosine kinase inhibitor decreases aberrant tyrosinephosphorylation of a transmembrane glycoprotein, such as thetransmemberane glycoprotein Protein Zero-Related (PZR). In anotherembodiment, the low-dosage tyrosine kinase inhibitor provides ananti-fibrotic effect in cardiac tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments, which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1A is an illustration of a proteomic analysis of differentiallytyrosyl-phosphorylated proteins in hearts of Ptpn11^(D61G/+) mice.Classification of hypo- and hyper-tyrosylphosphorylated proteins in thehearts of Ptpn11^(D61G/+) mice.

FIG. 1B is graph showing log 2-transformed values for the ratio of eachphosphotyrosine-containing peptide in wild-type and Ptpn11^(D61G/+)mouse hearts.

FIG. 1C is a heat map of differentially hyper-tyrosylphosphorylatedpeptides (the site of phosphorylation is identified by MS inparentheses).

FIG. 1D is a panel of images of extracted ion chromatogram and peptidesequence of PZR-containing tyrosine 242 (upper panels) and tyrosine 264(lower panels) by differential proteomics.

FIG. 1E shows amino acid sequences of the protein zero-related (PZR) Cterminus in different vertebrates. Consensus sequences forimmunoreceptor tyrosine-based inhibitory motif (ITIM; S/I/V/LXYXXI/V/L)are indicated in boldface, and tyrosine residues are marked red with theappropriate amino acid numbering. Sequences are shown for the PZR Cterminus from Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus,Canis lupus familiaris, Danio rerio, Gallus gallus.

FIG. 2A is a panel of blots showing the characterization of PZR tyrosylphosphorylation. C2C12 cells were cotransfected with empty vector oractivated glutathione S-transferase (GST)-Shp2E76A and either emptyvector (vector), wild-type human PZR (WT), or PZR mutated at tyrosine241 (Y241F), tyrosine 263 (Y263F), or both (2YF). Cell lysates wereimmunoblotted with anti-pPZR (Y241 or Y263), -PZR, or -Shp2 antibodies.

FIG. 2B is a panel of blots showing the characterization of PZR tyrosylphosphorylation. HEK-293 cells co-transfected with empty vector (Vec) oractivated Shp2E76A and either empty vector (vector), wild-type zebrafishPZR (WT), or PZR mutated at tyrosine 236 (Y241F), tyrosine 258 (Y263F),or both (2YF). Cell lysates were immunoblotted with anti-pPZR(Y241 orY263), PZR, or Shp2 antibodies. ERK1/2 was used as a loading control.

FIG. 2C is a panel of blots showing the characterization of PZR tyrosylphosphorylation. HUVECs were infected with adenoviruses expressingeither GFP as a control, wild-type Shp2, or Shp2E76A. Cell lysates wereimmunoblotted with anti-pPZR(Y241 or Y263), anti-total PZR, andanti-Shp2 antibodies.

FIG. 2D is a panel of blots showing the characterization of PZR tyrosylphosphorylation. HEK-293 cells were transiently transfected with emptyvector, wild-type Shp2 (WT), or the indicated Shp2 mutants (activatedShp2, E76A; Noonan syndrome (NS) mutant, N308D; or Noonan syndrome withmultiple lentigines (NSML) mutants, Y279C and T468M). Cell lysates wereimmunoblotted with anti-pPZR(Y241 or Y263), -PZR, and -Shp2 antibodies.ERK1/2 was used as a loading control.

FIG. 2E is a panel of blots showing the characterization of PZR tyrosylphosphorylation. HEK-293T cells were transfected with HA-taggedzebrafish PZR with empty vector, wild-type Shp2, Shp2D61G (NS mutant),or Shp2A462T (NSML mutant). Cell lysates were immunoprecipitated withanti-HA antibodies, and immune complexes were immunoblotted withanti-Shp2 and anti-HA antibodies. Whole-cell lysates (WCL) were blottedwith anti-pPZR(Y241 and Y263), -Shp2, and -HA antibodies.

FIG. 3A shows PZR tyrosyl phosphorylation in the hearts ofPtpn11^(D61G/+) mice. The heart was isolated from 5-week-old WT andPtpn11^(D61G/+) mice. Tissue lysates were immunoblotted with pPZR(Y263)and total PZR antibodies. Phosphorylation of tyrosine 264 in PZRrepresents n=5 per genotype. All data are means±standard errors of themeans (SEM). **, P<0.01.

FIG. 3B shows PZR tyrosyl phosphorylation in the cortex ofPtpn11^(D61G/+) mice. The cortex was isolated from 5-week-old WT andPtpn11^(D61G/+) mice. Tissue lysates were immunoblotted with pPZR(Y263)and total PZR antibodies. Phosphorylation of tyrosine 264 in PZRrepresents n=5 per genotype. All data are means±standard errors of themeans (SEM). ***, P<0.001.

FIG. 3C shows PZR tyrosyl phosphorylation in the heart ofPtpn11^(Y279C/+). The heart was isolated from 8-week-old WT andPtpn11^(Y279C/+) mice. Tissue lysates were immunoblotted with pPZR(Y263)and total PZR antibodies. Phosphorylation of tyrosine 264 in PZRrepresents n=5 per genotype. All data are means±standard errors of themeans (SEM). *, P<0.05.

FIG. 3D shows PZR tyrosyl phosphorylation in the cortex ofPtpn11^(Y279C/+) mice. The cortex was isolated from 8-week-old WT andPtpn11^(Y279C/+) mice. Tissue lysates were immunoblotted with pPZR(Y263)and total PZR antibodies. Phosphorylation of tyrosine 264 in PZRrepresents n=5 per genotype. All data are means±standard errors of themeans (SEM). ***, P<0.001.

FIG. 4A shows PZR tyrosyl phosphorylation in the liver ofPtpn11^(D61G/+) mice. The liver was isolated from 5-week-old wild-typeand Ptpn11^(D61G/+) mice. Tissue lysates were immunoblotted withanti-pPZR(Y263) and -total PZR antibodies. Densitometric analysis of thephosphorylation levels of tyrosine 263 in PZR was performed, and theresults represent the means±SEM from 5 mice per genotype.

FIG. 4B is an image and a graph showing PZR tyrosyl phosphorylation inthe kidney of Ptpn11^(D61G/+) mice. The kidney was isolated from5-week-old wild-type and Ptpn11^(D61G/+) mice. Tissue lysates wereimmunoblotted with anti-pPZR(Y263) and -total PZR antibodies.Densitometric analysis of the phosphorylation levels of tyrosine 263 inPZR was performed, and the results represent the means±SEM from 5 miceper genotype. **, P<0.01 (WT versus NS).

FIG. 4C is an image and a graph showing PZR tyrosyl phosphorylation inthe spleen of Ptpn11^(D61G/+) mice. The spleen was isolated from5-week-old wild-type and Ptpn11^(D61G/+) mice. Tissue lysates wereimmunoblotted with anti-pPZR(Y263) and -total PZR antibodies.Densitometric analysis of the phosphorylation levels of tyrosine 263 inPZR was performed, and the results represent the means±SEM from 5 miceper genotype. ***, P<0.001 (WT versus NS).

FIG. 5A is a panel of immunoblots showing ERK and Akt phosphorylation inthe heart of Ptpn11^(D61G/+) mice. The hearts were isolated from5-week-old wild-type and Ptpn11^(D61G/+) mice (A and B). Tissue lysateswere subjected to immunoblotting with anti-Shp2, -pERK1/2, -totalERK1/2, -pAkt, and -Akt antibodies. The results represent densitometricanalyses of the means±SEM for pERK1/2 and pAkt from 5 mice per genotype.

FIG. 5B is a panel of images showing ERK and Akt phosphorylation in thecortex of Ptpn11^(D61G/+) mice. The cortex was isolated from 5-week-oldwild-type and Ptpn11^(D61G/+) mice. Tissue lysates were subjected toimmunoblotting with anti-Shp2, -pERK1/2, -total ERK1/2, -pAkt, and -Aktantibodies. The results represent densitometric analyses of themeans±SEM for pERK1/2 and pAkt from 5 mice per genotype.

FIG. 5C is a panel of images showing ERK and Akt phosphorylation in theheart of Ptpn11^(Y279C/+) mice. The hearts were isolated from 8-week-oldwild-type and Ptpn11^(Y279C/+) mice. Tissue lysates were subjected toimmunoblotting with anti-Shp2, -pERK1/2, -total ERK1/2, -pAkt, and -Aktantibodies. The results represent densitometric analyses of themeans±SEM for pERK1/2 and pAkt from 5 mice per genotype.

FIG. 5D is a panel of images showing ERK and Akt phosphorylation in thecortex of Ptpn11^(Y279C/+) mice. The cortex was isolated from 8-week-oldwild-type and Ptpn11^(Y279C/+) mice. Tissue lysates were subjected toimmunoblotting with anti-Shp2, -pERK1/2, -total ERK1/2, -pAkt, and -Aktantibodies. The results represent densitometric analyses of themeans±SEM for pERK1/2 and pAkt from 5 mice per genotype.

FIG. 6A is a panel of blots showing the effect of Src family kinases onPZR Y241 and Y263 phosphorylation. HEK-293 cells were transientlytransfected with the indicated Shp2 mutants and treated with eitherdimethyl sulfoxide (DMSO) as a control or 5 μM SU6656. Cell lysates wereimmunoblotted with anti-Shp2, pSrc(Y416), Src, pPZR(Y241 or Y263), andtotal PZR antibodies. ERK1/2 was used as a loading control.

FIG. 6B is a panel of blots showing the effect of Src family kinases onPZR Y241 and Y263 phosphorylation. NIH 3T3 cells were infected with theadenoviruses expressing either GFP as a control or a constitutivelyactive Shp2E76A, in the presence of DMSO, PP2, or SU6656 at theindicated concentration. Cell lysates were immunoblotted with anti-Shp2,pSrc(Y416), Src, pPZR(Y241 or Y263), and total PZR antibodies. ERK1/2was used as a loading control.

FIG. 7A is a blot showing that Src kinase mediated NS- orNSML-Shp2-induced PZR hyper-tyrosyl phosphorylation. SYF cells(Src^(−/−) Fyn^(−/−) Yes^(−/−) MEFs) and Src cells (SYF cells expressingwild-type Src) were infected with adenoviruses expressing either GFP orShp2E76A. Cell lysates were immunoblotted with anti-Shp2, pSrc (Y416),pPZR (Y241 or Y263), PZR, and Src antibodies. ERK1/2 was used as aloading control.

FIG. 7B is a blot showing that Src kinase mediated NS- orNSML-Shp2-induced PZR hyper-tyrosyl phosphorylation. SYF cells weretransiently transfected with wild-type c-Src or kinase-deadc-SrcK295R/Y527F (KR/YF) and infected with adenoviruses expressingeither GFP, wild-type Shp2, or Shp2E76A. Cell lysates were immunoblottedwith anti-Shp2, pSrc (Y416), pPZR (Y241 or Y263), PZR, and Srcantibodies. ERK1/2 was used as a loading control.

FIG. 7C is a blot showing that Src kinase induced PZR hyper-tyrosylphosphorylation. HEK-293 cells were transfected with Flag-tagged humanPZR. The cell lysates were immunoprecipitated with anti-Flag antibody.The immunoprecipitates were subjected to in vitro Src kinase assay withSrc recombinant protein. The reaction products were immunoblotted withanti-pPZR (Y241 or Y263) antibodies.

FIG. 7D is a blot showing that Src family kinase induced PZRhyper-tyrosyl phosphorylation. HEK-293 cells were cotransfected withconstitutively active Src mutant and either HA-tagged wild-typezebrafish PZR (WT), PZR mutated at tyrosine 236 (Y241F), tyrosine 258(Y263F), or both (2YF). Cell lysates were immunoblotted with anti-pPZR(Y241 or Y263), and HA antibodies.

FIG. 8A is a blot and graph showing enhanced Src complex formation withNS/NSML-associated Shp2 mutants. HEK-293 cells were transientlytransfected either with the Shp2 WT and the indicated Shp2 mutants. Celllysates were immunoprecipitated (IP) with anti-c-Src antibodies, andimmune complexes were immunoblotted (IB) with anti-Shp2 and -Srcantibodies. The graph represents the means±SEM of the densitometricanalysis from three independent experiments. Statistical significancewas derived using a Dunnett's test comparing Shp2 mutants with the WT.*, P<0.05; **, P<0.01.

FIG. 8B is an image of a blot showing enhanced Src complex formationwith NS/NSML-associated Shp2 mutants and PZR. HEK-293 cells werecotransfected with the Shp2 WT or E76A or Y279C mutant and either emptyvector (vector), WT human PZR, or the PZR 2YF mutant. Cell lysates wereimmunoblotted with anti-pPZR(Y241 or Y263) or Shp2 antibodies. ERK1/2was used as a loading control. Immune complexes were immunoblotted withanti-Src, -Shp2, and -PZR antibodies.

FIG. 9 is an illustration of the model for the effects of NS and NSMLmutants on PZR tyrosyl phosphorylation.

FIG. 10 is a panel of images showing dosing of dasatinib. MalePtpn11D61G/+ mice were injected i.p. with dasatinib at the indicateddose or DMSO control, 24h later mice were sacrificed, heart tissueharvested and immunoblotted using the indicated antibodies.

FIG. 11A is an illustration showing the pre-natal dosing regimen fordasatinib in NS mouse model. Dasatinib was administered daily topregnant mothers between the time of when animals were in utero at E7.5until 9 days after birth (P9). At post-natal day 10 (P10), mice receiveddasatinib directly by daily injections for 46 days (P56). Mice wereevaluated for cardiac function at P42 and P56.

FIG. 11B is an illustration showing the post-natal dosing regimen fordasatinib in NS mouse model. Dasatinib was administered to mice startingat P10 daily for 32 days (P42) and discontinued for 14 days (P56). Micewere evaluated for cardiac function at P42 and P56.

FIG. 12A is a panel of graphs showing dasatinib-treated pre-natally inNS mice improves cardiac function. Pregnant mice were injected i.p. withdasatinib (0.1 mg/kg) according to protocol described herein. The numberof mice for each group is indicated. Statistical significance isindicated by; *; P<0.05, **; P<0.01, ***; P<0.001 by two-way ANOVA test.LV vol;s—Left ventricular volume in systole, LV vol;d—Left ventricularvolume in diastole, FS—Fractional shortening and EF—Ejection fraction.

FIG. 12B is a panel of graphs showing dasatinib-treated in post-natal NSmice improves cardiac function. NS mice starting at P10 were injectedi.p. with Disatinib (0.1 mg/kg) according to the protocol describedherein. Mice were evaluated for cardiac function after 32 days oftreatment. The number of mice for each group is indicated. Statisticalsignificance is indicated by; *; P<0.05, **; P<0.01, ***; P<0.001 bytwo-way ANOVA test. LV vol;s—Left ventricular volume in systole, LVvol;d—Left ventricular volume in diastole, FS—Fractional shortening andEF—Ejection fraction.

FIG. 12C is a panel of graphs showing preserved improvement of cardiacfunction after cessation of dasatinib treatment. NS mice starting at P10were injected i.p. with dasatinib (0.1 mg/kg) according to the protocolshown in FIG. 1B. Mice that had received dasatinib for 32 days werere-evaluated for cardiac function two weeks later. The number of micefor each group is indicated. Statistical significance is indicated by;*; P<0.05, **; P<0.01, ***; P<0.001 by two-way ANOVA test. LV vol;s—Leftventricular volume in systole, LV vol;d—Left ventricular volume indiastole, FS—Fractional shortening and EF—Ejection fraction.

FIG. 13, comprising FIGS. 13a-13l , is a panel of images showing c-Srckinase is a putative target for Noonan syndrome. FIG. 13a is a schematicdiagram of human Shp2 wild type full-length, N+C-SH2 and PTP domainconstructs. FIG. 13b is an image showing detection of Myc-Srcfull-length co-transfected with Flag-Shp2 full-length, N+C or PTP domaininto HEK-293T cells. Protein-protein interactions were determined byimmunoprecipitation. FIG. 13c is an image showing detection of purifiedGST-SH3 domain of c-Src incubated with purified His-tagged PTP domain ofShp2 overnight at 4° C. Proteins were immobilized with GST-Sepharosebeads and separated by SDS-PAGE. His-PTP domain was detected in GSTcomplexes by immunoblotting with anti-His antibodies. FIG. 13d is animage showing the phosphorylation levels of Src (Y416). Mouse embryonicfibroblasts (MEFs) from Ptpn11^(D61G/+) mice incubated with dasatinib atthe indicated concentrations for 18 hr. Whole cell lysates wereimmunoblotted with anti-p-Src (Y416), Src, p-ERK1/2 and ERK1/2antibodies. Tyrosyl-phosphorylation of PZR was determined withphospho-specific PZR antibodies and the molecular interaction betweenPZR and Shp2 was determined by immunoprecipitation. FIG. 13e is a graphshowing phosphorylation levels of Src (Y416). FIG. 13f is a graphshowing phosphorylation levels of ERK1/2. FIG. 13g is a graph showingthe amounts of PZR phosphorylation at tyrosine 241. FIG. 13h is a graphshowing the amounts of tyrosine 263 analyzed by densitometry. FIG. 13iis an image showing heart tissue immunoblotted with anti-Shp2, p-ERK1/2,ERK1/2, p-Src (Y416), Src, p-PZR (Y263) and PZR antibodies. 3-week-oldWT and Ptpn11^(D61G/+) mice were intraperitoneally injected with vehicleor dasatinib (0.1, 0.5 or 1.0 mg/kg). Heart tissue was isolated after 18hr and tissue lysates were immunoblotted. FIG. 13j is a graph showingtyrosyl phosphorylation of ERK1/2. FIG. 13k is a graph showing tyrosylphosphorylation of Src. FIG. 13l is a graph showing tyrosylphosphorylation of PZR. All data present mean±SEM. *, p<0.05; **,p<0.01; ***, p<0.001 denote significance compared with the vehicletreated Ptpn11^(D61G/+) MEFs (e-h) or heart tissues (j−1) (n=3 for eachcondition; One-way ANOVA test). WCL: whole cell lysates, IP:immunoprecipitation, D3: immunoblotting.

FIG. 14, comprising FIGS. 14a-14n , is a panel of images showing thatdasatinib improves cardiac functions of Ptpn11^(D61G/+) mice. FIG. 14ais a schematic diagram of prenatal dasatinib administration intoPtpn11^(D61G/+) mice. FIG. 14b is a panel of images showingrepresentative echocardiographic images of vehicle- or dasatinib-treatedWT and Ptpn11^(D61G/+) mice at P42. FIG. 14c is a graph showing thepercentage of ejection fraction (EF) measured from echocardiogram atP42. FIG. 14d is a panel of images showing representativeechocardiographic images of vehicle- or asatinib-treated WT andPtpn11^(D61G/+) mice at P56. FIG. 14e is a graph showing the percentageof ejection fraction (EF) measured from echocardiogram at P56. FIG. 14fis a schematic diagram of prenatal dasatinib administration intoPtpn11^(D61G/+) mice. FIG. 14g is a panel of images showingrepresentative echocardiographic images of vehicle- or dasatinib-treatedWT and Ptpn11^(D61G/+) mice at P42. FIG. 14h is a graph showing thepercentage of ejection fraction (EF) was measured from echocardiogram atP42. FIG. 14i is a panel of images showing representativeechocardiographic images of vehicle- or dasatinib-treated WT andPtpn11^(D61G/+) mice at P56. FIG. 14j is a graph showing the percentageof ejection fraction (EF) was measured from echocardiogram at P56. FIG.14k is a graph showing arterial systolic pressure from an invasivehemodynamic study of postnatal vehicle- or dasatinib-treated WT andPtpn11^(D61G/+) mice at P56. FIG. 14l is a graph showing the diastolicpressure of postnatal vehicle- or dasatinib-treated WT andPtpn11^(D61G/+) mice at P56. FIG. 14m is a graph showing the meanarterial pressure (MAP) of postnatal vehicle- or dasatinib-treated WTand Ptpn11^(D61G/+) mice at P56. FIG. 14n is a graph showing the leftventricle blood pressure (LV pressure) of postnatal vehicle- ordasatinib-treated WT and Ptpn11^(D61G/+) mice at P56. All data presentmean±SEM. *, p<0.05; **, p<0.01; ***, p<0.001. (n=10-16 for each group;Two-way ANOVA test).

FIG. 15, comprising FIG. 15a-15j , is a panel of images showingcardiomyopathy and cardiac fibrosis in Ptpn11^(D61G/+) mice rescued bydasatinib. FIG. 15a is a graph showing heart weight measured frompostnatal vehicle- or dasatinib-treated WT and Ptpn11^(D61G/+) mice atP56 (n=8-9 for each group). FIG. 15b is a graph showing heart weight(H.W.) to body weight (B.W.) ratio measured from postnatal vehicle- ordasatinib-treated WT and Ptpn11^(D61G/+) mice at P56 (n=8-9 for eachgroup). FIG. 15c is a panel of images showing the representative imagesof Masson's trichrome stained longitudinal sections of heart frompostnatal vehicle- or dasatinib-treated WT and Ptpn11^(D61G/+) mice atP56 (bar=2 mm). FIG. 15d is a panel of images showing Masson's trichromestain images of left ventricle from postnatal vehicle- ordasatinib-treated WT and Ptpn11^(D61G/+) mice at P56 (bar=200 μm). FIG.15e is a graph showing relative expression of fibrosis marker gene,Col1a2. Total heart RNA was isolated from postnatal vehicle- ordasatinib-treated WT and Ptpn11^(D61G/+) mice (P56). FIG. 15f is a graphshowing relative expression of fibrosis marker gene, Col3a1. FIG. 15g isa graph showing relative expression of cardiac fetal gene, Myh6 (αMHC).FIG. 15h is a graph showing relative expression of cardiac fetal gene,Myh7 (βMHC). FIG. 15i is a graph showing relative expression of cardiacfetal gene, Anf. FIG. 15j is a graph showing relative expression ofcardiac fetal gene, Bnp. The genes were measured by quantitative RT-PCR(n=6 for each group). All data present mean±SEM. *, p<0.05; **, p<0.01;***, p<0.001. (Two-way ANOVA test).

FIG. 16, comprising FIGS. 16a -FIG. 16g , is a panel of images showingcardiomyocytes from dasatinib treated Ptpn11^(D61G/+) mice exhibitednormal Ca²⁺ signaling during excitation-contraction coupling. FIG. 16ais an image showing Ca²⁺ excitation-contraction coupling measured incardiomyocytes isolated from postnatal vehicle- or dasatinib-treated WTand Ptpn11^(D61G/+) mice. Representative traces of cardiomyocytedynamics for calcium transient traces (top) and their correspondingsarcomere leng shortening traces (bottom). FIG. 16b is a graph showing asummary of the data of relative calcium release (R_(mag) Ca²⁺). FIG. 16cis a graph showing the fraction of sarcomere shortening (n=111-162 cellsfrom 3 mice for each group). FIG. 16d is an image showing heart tissueimmunoblotted with anti-SERCA2A, Troponin I (tTnI), Troponin T (tTnT)and Tubulin antibodies. The heart tissue was isolated from postnatalvehicle- or dasatinib-treated WT and Ptpn11^(D61G/+) mice and tissuelysates were immunoblotted. FIG. 16e is a graph showing SERCA2Aexpression. FIG. 16f is a graph showing Troponin I expression. FIG. 16gis a graph showing Troponin T. Expression was statistically assessedafter normalization with tubulin (n=6 for each group). All data presentmean±SEM. *, p<0.05; **, p<0.01; ***, p<0.001 (Two-way ANOVA test).

FIG. 17a-17g , comprising FIGS. 17a-17g , is a panel of images showingthe effects of Dasatinib on NS signaling in vitro. Mouse embryonicfibroblasts (MEFs) from Ptpn11^(D61G/+) mice were incubated withDasatinib for 18 hr. Whole cell lysates were immunoblotted withanti-Shp2, p-Src (Y416), Src, p-Raf1 (Y341), Raf1, p-MEK1/2, MEK1/2,p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, p38, p-Akt and Akt antibodies (FIG.17a ). The phosphorylation levels of Raf1(Y341) (FIG. 17b ), MEK1/2(FIG. 17c ), JNK (FIG. 17d ), and Akt(S473) (FIG. 17e ) werestatistically assessed. All data present mean±SEM. *, p<0.05; **,p<0.01; ***, p<0.001 denotes significance compared with the vehicletreated Ptpn11^(D61G/+) MEFs (n=3 for each condition; One-way ANOVAtest). Mouse embryonic fibroblasts (MEFs) from Ptpn11^(D61G/+) mice wereincubated with STI-571 (FIG. 17f ) or Shp2 inhibitor (FIG. 17g ) for 18hr. Tyrosyl-phosphorylation of PZR was determined with phospho-specificPZR antibodies.

FIG. 18, comprising FIGS. 18a-18f , is a panel of images showing theeffects of dasatinib on NS signaling in vivo. 3-weeks-old WT andPtpn11^(D61G/+) mice were intraperitoneally injected with vehicle ordasatinib (0.1, 0.5 or 1.0 mg/kg). FIG. 18a is an image showing hearttissue immunoblotted with p-Raf1 (Y341), Raf1, p-MEK1/2, MEK1/2, p-INK,INK, p-p38, p38, p-Akt (S473) and Akt antibodies. Heart tissue wasisolated after 18 hr and tissue lysates were immunoblotted. FIG. 18b isa graph showing the phosphorylation levels of Raf1(Y341). FIG. 18c is agraph showing the phosphorylation levels of MEK1/2. FIG. 18d is a graphshowing the phosphorylation levels of JNK. FIG. 18e is a graph showingthe phosphorylation levels of p38. FIG. 18f is a graph showing thephosphorylation levels of Akt(S473). All data present mean±SEM. *,p<0.05; **, p<0.01; ***, p<0.001 denotes significance compared with thevehicle treated Ptpn11^(D61G/+) mice (n=3 for each condition; One-wayANOVA test).

FIG. 19, comprising FIGS. 19a-19e , is a panel of images showingpostnatal dasatinib administration did not improve whole body growth inPtpn11^(D61G/+) mice. FIG. 19a is a graph showing growth curves ofpostnatal vehicle- or dasatinib-treated WT and Ptpn11^(D61G/+) mice.Differences within treatment groups were significant (p<0.001, Two-wayANOVA test) from 3-weeks to 8-weeks old. FIG. 19b is a graph showingbody weight measured at P42. FIG. 19c is a graph showing body lengthmeasured at P42. FIG. 19d is a graph showing body weight measured atP56. FIG. 19c is a graph showing body length measured at P42. FIG. 19eis a graph showing body length measured at P56. All data presentmean±SEM. *, p<0.05; **, p<0.01; ***, p<0.001 (n=10-16 for each group;Two-way ANOVA test).

FIG. 20, comprising FIGS. 20a-20k , is a panel of images showing thatthe facial dysmorphic features were not changed in dasatinib treated inPtpn11^(D61G/+) mice. FIG. 20a is a panel of representative images ofthe skull and the lower jaw from postnatal vehicle- or dasatinib-treatedWT and Ptpn11D^(61G/+) mice at P56. FIG. 20b is a graph showingmeasurements obtained from the skull length from vehicle- ordasatinib-treated WT and Ptpn11D^(61G/+) mice at P42. FIG. 20c is agraph showing measurements obtained from the skull width from vehicle-or dasatinib-treated WT and Ptpn11D^(61G/+) mice at P42. FIG. 20d is agraph showing measurements obtained from the ratio of skull length toskull width from vehicle- or dasatinib-treated WT and Ptpn11D^(61G/+)mice at P42. FIG. 20e is a graph showing measurements obtained from theintercantal distance (ICD) from vehicle- or dasatinib-treated WT andPtpn11D^(61G/+) mice at P42. FIG. 20f is a graph showing measurementsobtained from the lower jaw length from vehicle- or dasatinib-treated WTand Ptpn11D^(61G/+) mice at P42. FIG. 20g is a graph showingmeasurements obtained from the skull length from vehicle- ordasatinib-treated WT and Ptpn11D^(61G/+) mice at P56. FIG. 20h is agraph showing measurements obtained from the skull width from vehicle-or dasatinib-treated WT and Ptpn11D^(61G/+) mice at P56. FIG. 20i is agraph showing measurements obtained from the ratio of skull length toskull width from vehicle- or dasatinib-treated WT and Ptpn11D^(61G/+)mice at P56. FIG. 20j is a graph showing measurements obtained from theintercantal distance (ICD) from vehicle- or dasatinib-treated WT andPtpn11D^(61G/+) mice at P56. FIG. 20k is a graph showing measurementsobtained from the lower jaw length from vehicle- or dasatinib-treated WTand Ptpn11D^(61G/+) mice at P56. All data present mean±SEM. *, p<0.05;**, p<0.01;***, p<0.001. (n=10-16 for each group; Two-way ANOVA test).

FIG. 21, comprising FIGS. 21a-21e , is a panel of images showing thatpostnatal dasatinib treatment did not rescue the splenomegaly phenotypein Ptpn11^(D61G/+) mice. FIG. 21a is a panel of representative H&Estained histological images of the spleen from postnatal vehicle- ordasatinib-treated 8-week-old WT and Ptpn11D^(61G/+) mice at P56 (bar=200μm). FIG. 21b is a graph showing the spleen weight measured fromvehicle- or dasatinib-treated WT and Ptpn11D^(61G/+) mice at P42. FIG.21c is a graph showing the ratio of spleen weight to body weightmeasured from vehicle- or dasatinib-treated WT and Ptpn11D^(61G/+) miceat P42. FIG. 21d is a graph showing the spleen weight measured fromvehicle- or dasatinib-treated WT and Ptpn11D^(61G/+) mice at P56. FIG.21e is a graph showing the ratio of spleen weight to body weightmeasured from vehicle- or dasatinib-treated WT and Ptpn11D^(61G/+) miceat P56. All data represent mean±SEM. *, p<0.05; ***, p<0.001. (n=7-10for each group; Two-way ANOVA test).

FIG. 22, comprising FIGS. 22a-22c , is a panel of images showingdasatinib does not induce liver damage. FIG. 22a is a panel ofrepresentative H&E stained histological images of the liver frompostnatal vehicle- or dasatinib-treated 8-week-old WT andPtpn11D^(61G/+) mice at P56 (bar=200 μm). FIG. 22b is a graph showingthe enzymatic activities of alanine aminotransferase (ALT) in serummeasured from vehicle- or dasatinib-treated WT and Ptpn11D^(61G/+) miceat P42. FIG. 22c is a graph showing the enzymatic activities of alanineaminotransferase (ALT) in serum measured from vehicle- ordasatinib-treated WT and Ptpn11D^(61G/+) mice at P56. All data presentmean±SEM. *, p<0.05; ***, p<0.001. (n=7-10 for each group; Two-way ANOVAtest).

FIG. 23 is an image showing the molecular interaction between SH3 domainof Src and PTP domain of Shp2. Purified GST-tagged SH3 domain of c-Srcwas incubated with purified His-tagged PTP domain of Shp2 overnight at4° C. Proteins were immobilized with GST-Sepharose beads and separatedby SDS-PAGE. His-PTP domain was detected in GST complexes byimmunoblotting with anti-His antibodies.

FIG. 24, comprising FIGS. 24a-24b , is a panel of images showing PZRhyperphosphorylation induced by NS-Shp2 is not dependent on Shp2phosphatase or c-Abl kinase activities. Mouse embryonic fibroblasts(MEFs) from Ptpn11^(D61G/+) mice were incubated with STI-571 (FIG. 24a )or Shp2 inhibitor (FIG. 24b ) for 18 hr. Tyrosyl-phosphorylation of PZRwas determined with phospho-specific PZR antibodies.

FIG. 25, comprising FIGS. 25a-25f , is a panel of graphs showing theimprovement in molecular markers of cardiomyopathy and fibrosis in NSML(Y279C/+) mice following dasatinib treatment. Total heart RNA wasisolated from postnatal vehicle- or dasatinib-treated (0.1 mg/kg/day) WTand Ptpn11^(Y279C/+) mice (P42). Fibrosis marker genes, Col1a2 (FIG. 25a) and Col3a1 (FIG. 25b ), markers of cardiomyopathy ANP (FIG. 25c ) andcardiac fetal genes, Myh6 (αMHC) (FIG. 25d ), Myh7 (bMHC) (FIG. 25e ),and Myh6/Myh7 ratio (FIG. 25f ) were measured by quantitative RT-PCR(n=6 for each group). All data represent mean±SEM. *, p<0.05; **,p<0.01; ***, p<0.001. (Two-way ANOVA test).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein may be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

As used herein, the articles “a” and “an” are used to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein when referring to a measurable value such as an amount, atemporal duration, and the like, the term “about” is meant to encompassvariations of ±20% or within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.5%, 0.1%, 0.05%, or 0.01% of the specified value, as such variationsare appropriate to perform the disclosed methods. Unless otherwise clearfrom context, all numerical values provided herein are modified by theterm about.

The phrase “aberrant protein tyrosine phosphorylation” refers tohyperphosphorylation or hypophosphorylation of one or more targetproteins and/or abnormal protein kinase activity. In one embodiment, theaberrant protein tyrosine phosphorylation is compared to a control.

The term “cardiovascular disease or condition” refers to any disease orcondition which affects the cardiovascular system including, but notlimited to, nerve conduction disorders, thrombophilia, atherosclerosis,angina pectoris, hypertension, arteriosclerosis, myocardial infarction,congestive heart failure, cardiomyopathy, hypertension, arterial andvenous stenosis, valvular disease, myocarditis and arrhythmias.Conditions of cardiovascular disease also include, but are not limitedto, any clinical manifestation of a disease state associated with theheart and the central or peripheral arterial and venous vasculature. Forexample, said clinical manifestations include, but are not limited topain, weakness, high blood pressure, elevated plasma cholesterol,elevated plasma fatty acids, tachycardia, bradycardia, abnormalelectrocardiogram, external or internal bleeding, headache, dizziness,nausea and vomiting.

The term “cardiac function” refers to an activity of the heart orinteraction of cells or tissues in the heart to perform an activity.Examples of a cardiac function include, but are not limited to,myofibrilar organization, cardiomyocyte contractility, adequate deliveryof blood and nutrients to tissues required. Abnormal cardiac function(inadequate delivery of blood and nutrients to tissues]) can lead toproblems, such as but not limited to, blood pressure changes,thrombosis, electrocardiographic changes, arrhythmias, myocarditis,pericarditis, myocardial infarction, cardiomyopathy, hypertrophy,hypotrophy, cardiac failure (ventricular failure (left or right)),congestive heart failure, and cardiac arrest. An improvement of cardiacfunction may include an improvement, elimination or prevention of atleast one abnormal cardiac function, such as but not limited to,myofibrilar disorganization, abnormal cardiomyocyte contractility,cardiac fibrosis, abnormal blood pressure, excess blood pressurechanges, thrombosis, electrocardiographic changes, arrhythmias,myocarditis, pericarditis, myocardial infarction, cardiomyopathy, andcongestive heart failure.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

By “congenital heart disease” is meant a category of heart disease thatincludes abnormalities in cardiovascular structures that occur beforebirth.

By “effective amount” is meant the amount required to reduce or improveat least one symptom of a disease relative to an untreated patient. Theeffective amount of an active compound(s) used for therapeutic treatmentof a disease varies depending upon the manner of administration, theage, body weight, and general health of the subject.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

By “fragment” is meant a portion of a polynucleotide or nucleic acidmolecule. This portion contains, preferably, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acids. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80,90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000 or2500 (and any integer value in between) nucleotides. The fragment, asapplied to a nucleic acid molecule, refers to a subsequence of a largernucleic acid. A “fragment” of a nucleic acid molecule may be at leastabout 15 nucleotides in length; for example, at least about 50nucleotides to about 100 nucleotides; at least about 100 to about 500nucleotides, at least about 500 to about 1000 nucleotides, at leastabout 1000 nucleotides to about 1500 nucleotides; or about 1500nucleotides to about 2500 nucleotides; or about 2500 nucleotides (andany integer value in between).

By “low-dosage” is meant a therapeutically effective dosage that islower than dosages typically prescribed for indications other than heartdisease, congenital heart disease, heart failure or similar conditions.In one embodiment, the low-dosage is lower than a chemotherapeuticdosage. In another embodiment, the low-dosage is in the range of about200-fold lower than a chemotherapeutic dosage of a tyrosine kinaseinhibitor. In another embodiment, the low-dosage tyrosine kinaseinhibitor improves at least one cardiac function. Dasatinib has beenshown to be effective in preventing tumor incidence in mice at a dosageof ˜20 mg/kg (Kantarjian, H. et al. Dasatinib versus imatinib in newlydiagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 362,2260-2270 (2010)). The therapeutic effects of dasatinib in humans isreported to be ˜2 mg/kg, an equivalent dose of ˜24 mg/kg in mice (Yu, E.Y. et al. Phase II study of dasatinib in patients with metastaticcastration-resistant prostate cancer. Clinical cancer research: anofficial journal of the American Association for Cancer Research 15,7421-7428 (2009) and Apperley, J. F. et al. Dasatinib in the treatmentof chronic myeloid leukemia in accelerated phase after imatinib failure:the START a trial. J Clin Oncol 27, 3472-3479,doi:10.1200/JCO.2007.14.3339 (2009)). Doses of dasatinib as low as 0.1mg/kg (˜200-fold lower than therapeutic dose) were sufficient to treatCHD-associated cardiac disease.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation. A “purified” or“biologically pure” protein is sufficiently free of other materials suchthat any impurities do not materially affect the biological propertiesof the protein or cause other adverse consequences. That is, a nucleicacid or peptide is purified if it is substantially free of cellularmaterial, viral material, or culture medium when produced by recombinantDNA techniques, or chemical precursors or other chemicals whenchemically synthesized. Purity and homogeneity are typically determinedusing analytical chemistry techniques, for example, polyacrylamide gelelectrophoresis or high performance liquid chromatography. The term“purified” can denote that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. For a protein that canbe subjected to modifications, for example, phosphorylation orglycosylation, different modifications may give rise to differentisolated proteins, which can be separately purified.

“Pharmaceutically acceptable” refers to those properties and/orsubstances that are acceptable to the patient from apharmacological/toxicological point of view and to the manufacturingpharmaceutical chemist from a physical/chemical point of view regardingcomposition, formulation, stability, patient acceptance andbioavailability. “Pharmaceutically acceptable carrier” refers to amedium that does not interfere with the effectiveness of the biologicalactivity of the active ingredient(s) and is not toxic to the host towhich it is administered.

As used herein, the term “pharmaceutical composition” or“pharmaceutically acceptable composition” refers to a mixture of atleast one compound or molecule useful within the invention with apharmaceutically acceptable carrier. The pharmaceutical compositionfacilitates administration of the compound or molecule to a patient.Multiple techniques of administering a compound or molecule exist in theart including, but not limited to, intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound or molecule usefulwithin the invention within or to the patient such that it may performits intended function. Typically, such constructs are carried ortransported from one organ, or portion of the body, to another organ, orportion of the body. Each carrier must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation,including the compound useful within the invention, and not injurious tothe patient. Some examples of materials that may serve aspharmaceutically acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; surface activeagents; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations. As usedherein, “pharmaceutically acceptable carrier” also includes any and allcoatings, antibacterial and antifungal agents, and absorption delayingagents, and the like that are compatible with the activity of thecompound useful within the invention, and are physiologically acceptableto the patient. Supplementary active compounds may also be incorporatedinto the compositions. The “pharmaceutically acceptable carrier” mayfurther include a pharmaceutically acceptable salt of the compound ormolecule useful within the invention. Other additional ingredients thatmay be included in the pharmaceutical compositions used in the practiceof the invention are known in the art and described, for example inRemington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co.,1985, Easton, Pa.), which is incorporated herein by reference.

By “Protein Zero-Related” or “PZR,” also called “myelin proteinzero-like protein 1” or “MPZL1” is meant a protein that is animmunoglobulin superfamily cell surface protein. PZR contains twoimmunoreceptor tyrosine-based inhibition motifs (ITIMs) responsible forbinding to Shp2. When phosphorylated, PZR can specifically bind Shp2,resulting in the activation of the tyrosine phosphatase activity ofShp2. Once activated, the tyrosine phosphatase activity of Shp2 servesto dephosphorylate downstream substrates that propagate cell signals.Shp2 can also signal by acting as a scaffold or an adaptor proteinwhereby it recruits other molecules/activities to specific complexes.Shp2 can control signaling in both a catalytically-dependent andindependent manner.

One isoform of PZR, called PZR1b, lacks the ITIMs and has a dominantnegative effect upon full-length PZR and its recruitment of Shp2. Anexemplary PZR sequence includes human PZR found at GenBank Accession No.NM_001146191 and NP_001139663, or a fragment thereof, and the mouse PZRsequence found at NM_001001880 or NP_001001880, or a fragment thereof.Much of the information known about PZR relates to its role inadhesion-mediated cell signaling and cell migration. However, whetherPZR is involved in pathophysiological cell signaling remains unknown andsubsequently the validity of PZR as a target for any human disease hasnot yet been realized.

By “RASopathy” is meant a group of genetic syndromes caused by germlinemutations in genes that encode components or regulators of theRas/mitogen-activated protein kinase (MAPK) pathway. These syndromesinclude neurofibromatosis type 1, Noonan syndrome, Noonan syndrome withmultiple lentigines, capillary malformation-arteriovenous malformationsyndrome, Costello syndrome, cardio-facio-cutaneous syndrome, and Legiussyndrome. The Ras/MAPK pathway plays an essential role in regulating thecell cycle and cellular growth, differentiation, and senescence, all ofwhich are critical to normal development. Because of the commonunderlying Ras/MAPK pathway dysregulation, the RASopathies exhibitnumerous overlapping phenotypic features. These overlapping phenotypescan in some cases exist or be caused by mechanisms that operateindependently of MAPK itself. The PZR/Shp2 complex described herein liesupstream of Ras.

Noonan syndrome (NS) is an autosomal dominant disorder that occurs withan incidence of about 1:1,000-2,500 live births in the U.S. The cardiacdefects most often recognized in NS are pulmonary valve stenosis,atrial-septal defect, and hypertrophic cardiomyopathy, with the severityof each ranging from mild to life-threatening. Noonan syndrome withmultiple lentigines (NSML) is a rare autosomal dominant disorder with asimilar phenotype to NS, including a “Noonan-like” appearance as well asmultiple lentigines, electroconduction abnormalities, ocularhypertelorism, pulmonary valve stenosis, abnormal genitalia, retardationof growth, and deafness. NS-associated mutations result in increasedphosphatase activity. NSML-associated mutations result in decreasedphosphatase activity.

By “reference” is meant a standard or control. A “reference” is adefined standard or control used as a basis for comparison.

As used herein, “sample” or “biological sample” refers to anything,which may contain the cells of interest (e.g., cancer or tumor cellsthereof) for which the screening method or treatment is desired. Thesample may be a biological sample, such as a biological fluid or abiological tissue. In one embodiment, a biological sample is a tissuesample including pulmonary arterial endothelial cells. Such a sample mayinclude diverse cells, proteins, and genetic material. Examples ofbiological tissues also include organs, tumors, lymph nodes, arteriesand individual cell(s). Examples of biological fluids include urine,blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinalfluid, tears, mucus, amniotic fluid or the like.

The “Src family of tyrosine kinases” or “SFKs” are a family of enzymesthat catalyze the addition of phosphate groups on to tyrosine residuesof protein substrates. c-Src represents one member of the SFK family.

By “Src family tyrosine kinase inhibitor” is meant a molecule thatdecreases or prevents phosphorylation of tyrosine residues on Src familyprotein substrates. The Src family tyrosine kinase inhibitor can disrupttyrosyl phosphorylation, bind the tyrosine kinase or tyrosine residue,possibly with higher association efficiency than the tyrosine kinase orphosphate group, and/or prevent effective binding of the phosphate groupto the tyrosine residue to decrease or prevent phosphorylation. Srcfamily tyrosine kinase inhibitors include, but are not limited to, smallmolecule Src family tyrosine kinase inhibitors, Src family tyrosinekinase antagonists, neutralizing antibodies, and inhibitory peptidesand/or oligonucleotides. Examples of small molecule Src family tyrosinekinase inhibitors include but are not limited to A419259, AP23451,AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675,CU201, ENMD 2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765,PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656,TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228,altenusin, bosutinib, damnacanthal, dasatinib, herbimycin A, indirubin,neratinib, lavendustin A, pelitinib, piceatannol, saracatinib, SrcI1,and analogs thereof.

“Src homology 2 (SH2) domain-containing (SH2) protein tyrosinephosphatase-2” or “Shp2” is a member of the tyrosine-specific family ofprotein tyrosine phosphatases (PTPs). Shp2 is a tyrosine phosphatasethat catalyzes the tyrosine dephosphorylation of proteins. Mutations inthe human gene, PTPN11, have been found to cause about half of Noonansyndrome cases and about one tenth of NSML cases.

A “subject” or “patient,” as used therein, may be a human or non-humanmammal. Non-human mammals include, for example, livestock and pets, suchas ovine, bovine, porcine, canine, feline and murine mammals.Preferably, the subject is human.

The term “transmembrane glycoprotein” refers to a membrane protein thatspans the cell membrane. In one embodiment, the transmembraneglycoprotein includes immunoglobulin superfamily cell surface proteins,such as PZR.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or improving a disorder and/or symptom associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely ameliorated or eliminated.

By “tyrosine kinase inhibitor” is meant a molecule that decreases orprevents phosphorylation of tyrosine residues on protein substrates. Thetyrosine kinase inhibitor can disrupt tyrosyl phosphorylation, bind thetyrosine kinase or tyrosine residue, possibly with higher associationefficiency than the tyrosine kinase or phosphate group, and/or preventeffective binding of the phosphate group to the tyrosine residue todecrease or prevent phosphorylation. Tyrosine kinase inhibitors include,but are not limited to, small molecule tyrosine kinase inhibitors,tyrosine kinase antagonists, neutralizing antibodies, and inhibitorypeptides and/or oligonucleotides. Examples of small molecule tyrosinekinase inhibitors include but are not limited to afatinib, axitinib,bosutinib, cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib,dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib,lapatinib, lenvatinib, lestaurtinib, nilotinib, nintedanib, palbociclib,pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus,sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib,and vemurafenib.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

The recitation of an embodiment for a variable or aspect herein includesthat embodiment as any single embodiment or in combination with anyother embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

Compositions

It has been discovered that aberrant protein tyrosine phosphorylation,such as phosphorylation of Src family tyrosine kinases and theirsubstrates, is altered in subjects with cardiovascular disease. It hasalso been discovered that aberrant protein tyrosine phosphorylation isaltered in subject with certain diseases, such as RASopathies.Inhibition of tyrosine kinase activity treats heart disease and improvesat least one cardiac function. Inhibition also improves cardiovascularfunction in subjects with congenital heart defects associated with aRASopathy. The invention includes compositions that inhibit tyrosinekinases, such as Src family tyrosine kinases, to improve at least onecardiac function, thereby preventing or decreasing tyrosinephosphorylation. The invention includes, in one aspect, a compositioncomprising a low-dosage tyrosine kinase inhibitor, wherein thelow-dosage tyrosine kinase inhibitor decreases tyrosine phosphorylationand improves at least one cardiac function in a subject in need thereof.

In one embodiment, the low-dosage tyrosine kinase inhibitor decreasesaberrant tyrosine phosphorylation of a transmembrane glycoprotein, suchas a Src family tyrosine kinase and Protein Zero-Related (PZR).

In one embodiment, the amount of the low-dosage tyrosine kinaseinhibitor is in the range of about 25 fold to about 500 fold lower thana chemotherapeutic dosage of the tyrosine kinase inhibitor. In anotherembodiment, the amount of the low-dosage tyrosine kinase inhibitor is inthe range of about 25 fold to about 400 fold lower than achemotherapeutic dosage of the tyrosine kinase inhibitor. In anotherembodiment, the amount of the low-dosage tyrosine kinase inhibitor is inthe range of about 25 fold to about 300 fold lower than achemotherapeutic dosage of the tyrosine kinase inhibitor. In anotherembodiment, the amount of the low-dosage tyrosine kinase inhibitor is inthe range of about 35 fold to about 300 fold lower than achemotherapeutic dosage of the tyrosine kinase inhibitor. In anotherembodiment, the amount of the low-dosage tyrosine kinase inhibitor is inthe range of about 50 fold to about 300 fold lower than achemotherapeutic dosage of the tyrosine kinase inhibitor. In anotherembodiment, the amount of the low-dosage tyrosine kinase inhibitor is inthe range of about 100 fold to about 300 fold lower than achemotherapeutic dosage of the tyrosine kinase inhibitor. In anotherembodiment, the amount of the low-dosage tyrosine kinase inhibitor is inthe range of about 150 fold to about 250 fold lower than achemotherapeutic dosage of the tyrosine kinase inhibitor. In anotherembodiment, the amount of the low-dosage tyrosine kinase inhibitor is inthe range of about 175 fold to about 250 fold lower than achemotherapeutic dosage of the tyrosine kinase inhibitor. The low-dosagetyrosine kinase inhibitor can be about 25 fold, 30 fold, 35 fold, 40fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold, 80fold, 85 fold, 90 fold, 95 fold, 100 fold, 105 fold, 110 fold, 115 fold,120 fold, 125 fold, 130 fold, 135 fold, 140 fold, 145 fold, 150 fold,155 fold, 160 fold, 165 fold, 170 fold, 175 fold, 180 fold, 185 fold,190 fold, 195 fold, 200 fold, 205 fold, 210 fold, 215 fold, 220 fold,225 fold, 230 fold, 235 fold, 240 fold, 245 fold, 250 fold, 255 fold,260 fold, 265 fold, 270 fold, 275 fold, 280 fold, 285 fold, 290 fold,295 fold, 300 fold, and any fold change therebetween lower than achemotherapeutic dosage of the tyrosine kinase inhibitor. In someembodiments, the chemotherapeutic dosage of the tyrosine kinaseinhibitor is in the range of about 75 to about 170 mg/day or about 1.1to about 2.4 for a 70 kg adult.

In another embodiment, the low-dosage tyrosine kinase inhibitor isselected from the group consisting of afatinib, axitinib, bosutinib,cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib, dasatinib,erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib,lenvatinib, lestaurtinib, nilotinib, nintedanib, palbociclib, pazopanib,ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib,sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, andvemurafenib. In another embodiment, the composition comprises more thanone of the tyrosine kinase inhibitors disclosed herein.

In still another embodiment, the low-dosage tyrosine kinase inhibitor isa Src family tyrosine kinase inhibitor, such as but not limited to aninhibitor selected from the group consisting A419259, AP23451, AP23464,AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285,PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003,TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228, altenusin,bosutinib, damnacanthal, dasatinib, herbimycin A, indirubin, neratinib,lavendustin A, pelitinib, piceatannol, saracatinib, SrcI1, and analogsthereof. In another embodiment, the composition comprises at least oneSrc family tyrosine kinase inhibitor.

In another embodiment, the low-dosage tyrosine kinase inhibitor improvesat least one cardiac function. The cardiac function may include, but isnot limited to, myofibrilar organization, cardiomyocyte contractility,SERCA2A expression, and cardiac fibrosis. Abnormal cardiac function canlead to problems, such as but not limited to, blood pressure changes,thrombosis, electrocardiographic changes, arrhythmias, myocarditis,pericarditis, myocardial infarction, cardiomyopathy, cardiac failure(ventricular failure), congestive heart failure, and cardiac arrest. Animprovement of cardiac function may include an improvement, eliminationor prevention of at least one abnormal cardiac function, such as but notlimited to, myofibrilar disorganization, abnormal cardiomyocytecontractility, dysregulated SERCA2A expression, cardiac fibrosis,abnormal blood pressure, excess blood pressure changes, thrombosis,electrocardiographic changes, arrhythmias, myocarditis, pericarditis,myocardial infarction, cardiomyopathy, and congestive heart failure.

In yet another embodiment, the low-dosage tyrosine kinase inhibitorprovides an anti-fibrotic effect. Increased levels of fibroticcomponents in the myocardium has been associated with the progression ofheart failure. Tyrosine kinase inhibition at a low-dosage reduces theaccumulation of fibrotic components in the myocardium.

Compositions that decrease aberrant protein tyrosine phosphorylation arealso included in the invention. Certain diseases, such as cardiovasculardisease or conditions like congenital heart disease, are characterizedby aberrant protein tyrosine phosphorylation. Treatments that prevent ordecrease tyrosine phosphorylation of one or more transmembraneglycoproteins, such as protein zero-related or PZR, are thereforeincluded in the invention. In another aspect, the invention includes acomposition that is capable of decreasing aberrant protein tyrosinephosphorylation associated with cardiovascular disease or condition. Inyet another aspect, the invention includes a composition that is capableof decreasing aberrant protein tyrosine phosphorylation associated withcongenital heart disease. In still another aspect, the inventionincludes a composition that is capable of decreasing aberrant proteintyrosine phosphorylation associated with cardiovascular disease orcondition associated with a RASopathy.

Methods

The present invention also includes a method for preventing or treatinga cardiovascular disease or condition in a subject in need thereof. Asdescribed herein, inhibition of aberrant tyrosine phosphorylationprevents and/or treats the cardiovascular disease or condition.Administering a composition that includes a low-dosage of a tyrosinekinase inhibitor to a subject, such as a pediatric subject, in needthereof to decrease aberrant levels of tyrosine phosphorylation forpreventing or treating cardiovascular disease or condition.

In one aspect, the invention includes a method of treatingcardiovascular disease or condition having aberrant protein tyrosinephosphorylation in a subject, comprising administering a low-dosage of atyrosine kinase inhibitor to a subject in need thereof, wherein thetyrosine kinase inhibitor decreases aberrant levels of tyrosinephosphorylation and improves at least one cardiac function in thesubject.

In another aspect, the invention includes a method of treatingcongenital heart disease comprising administering a low-dosage of atyrosine kinase inhibitor to a subject in need thereof, wherein thetyrosine kinase inhibitor decreases aberrant levels of tyrosinephosphorylation and improves at least one cardiac function in thesubject.

In yet another aspect, the invention includes a method of treatingcardiovascular disease or condition associated with a RASopathy havingaberrant protein tyrosine phosphorylation comprising administering alow-dosage of a tyrosine kinase inhibitor to a subject in need thereof,wherein the tyrosine kinase inhibitor decreases aberrant levels ofProtein Zero-Related (PZR) tyrosyl phosphorylation and improves at leastone cardiac function in the subject.

In one embodiment, the cardiovascular disease or condition in the methoddescribed herein is congenital heart disease or a cardiovascular diseaseor condition associated with a RASopathy, such as but not limited to aRASopathy selected from the group consisting of Neurofibromatosis Type1, Noonan syndrome, Noonan syndrome with multiple lentigines (Leopardsyndrome), capillary malformation-arteriovenous malformation syndrome,Costello syndrome, cardio-facio-cutaneous syndrome, and Legius syndrome.

In another embodiment, the method includes administering the tyrosinekinase inhibitor to a subject that is a pediatric patient. The pediatricsubject can be less than 18 years of age. The pediatric subject can beless than 12 years of age. The pediatric subject can be less than 10, 9,8, 7, 6, 5, 4, 3, 2, and 1 years of age. In another embodiment, thesubject is a pediatric patient that is less than 12 years of age. In analternative embodiment, the method includes administering the tyrosinekinase inhibitor to a subject that is greater than 18 years of age.

In one embodiment, the method includes administering a low-dosagetyrosine kinase inhibitor that decreases aberrant tyrosinephosphorylation of a transmembrane glycoprotein, such as a Src familytyrosine kinase and Protein Zero-Related (PZR). In one embodiment, theaberrant levels of tyrosine phosphorylation comprise aberrant levels oftyrosine phosphorylated Protein Zero-Related (PZR).

In another embodiment, administering the low-dosage tyrosine kinaseinhibitor improves a cardiac function, such as but not limited to,myofibrilar organization, cardiomyocyte contractility, SERCA2Aexpression, and cardiac fibrosis. In another embodiment, an improvementof a cardiac function may include an improvement, elimination orprevention of at least one abnormal cardiac function, such as but notlimited to, myofibrilar disorganization, abnormal cardiomyocytecontractility, dysregulated SERCA2A expression, cardiac fibrosis,abnormal blood pressure, excess blood pressure changes, thrombosis,electrocardiographic changes, arrhythmias, myocarditis, pericarditis,myocardial infarction, cardiomyopathy, and congestive heart failure. Inyet another embodiment, administering the low-dosage tyrosine kinaseinhibitor provides an anti-fibrotic effect to the subject.

In one embodiment, the dose of the low-dosage tyrosine kinase inhibitorused in the method described herein is in the range of about 25 fold toabout 500 fold lower than a chemotherapeutic dosage of the tyrosinekinase inhibitor. In another embodiment, the amount of the low-dosagetyrosine kinase inhibitor is in the range of about 25 fold to about 400fold lower than a chemotherapeutic dosage of the tyrosine kinaseinhibitor. In another embodiment, the amount of the low-dosage tyrosinekinase inhibitor is in the range of about 25 fold to about 300 foldlower than a chemotherapeutic dosage of the tyrosine kinase inhibitor.In another embodiment, the amount of the low-dosage tyrosine kinaseinhibitor is in the range of about 35 fold to about 300 fold lower thana chemotherapeutic dosage of the tyrosine kinase inhibitor. In anotherembodiment, the amount of the low-dosage tyrosine kinase inhibitor is inthe range of about 50 fold to about 300 fold lower than achemotherapeutic dosage of the tyrosine kinase inhibitor. In anotherembodiment, the amount of the low-dosage tyrosine kinase inhibitor is inthe range of about 100 fold to about 300 fold lower than achemotherapeutic dosage of the tyrosine kinase inhibitor. In anotherembodiment, the amount of the low-dosage tyrosine kinase inhibitor is inthe range of about 150 fold to about 250 fold lower than achemotherapeutic dosage of the tyrosine kinase inhibitor. In anotherembodiment, the amount of the low-dosage tyrosine kinase inhibitor is inthe range of about 175 fold to about 250 fold lower than achemotherapeutic dosage of the tyrosine kinase inhibitor. The low-dosagetyrosine kinase inhibitor can be about 25 fold, 30 fold, 35 fold, 40fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold, 80fold, 85 fold, 90 fold, 95 fold, 100 fold, 105 fold, 110 fold, 115 fold,120 fold, 125 fold, 130 fold, 135 fold, 140 fold, 145 fold, 150 fold,155 fold, 160 fold, 165 fold, 170 fold, 175 fold, 180 fold, 185 fold,190 fold, 195 fold, 200 fold, 205 fold, 210 fold, 215 fold, 220 fold,225 fold, 230 fold, 235 fold, 240 fold, 245 fold, 250 fold, 255 fold,260 fold, 265 fold, 270 fold, 275 fold, 280 fold, 285 fold, 290 fold,295 fold, 300 fold, and any fold change therebetween lower than achemotherapeutic dosage of the tyrosine kinase inhibitor. In someembodiments, the chemotherapeutic dosage of the tyrosine kinaseinhibitor is in the range of about 75 to about 170 mg/day or about 1.1to about 2.4 for a 70 kg adult.

In yet another embodiment, the low-dosage tyrosine kinase inhibitor isselected from the group consisting of afatinib, axitinib, bosutinib,cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib, dasatinib,erlotinib, everolimus, gefitinib, ibrutinib, imatinib, lapatinib,lenvatinib, lestaurtinib, nilotinib, nintedanib, palbociclib, pazopanib,ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib,sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib, andvemurafenib. In another embodiment, the tyrosine kinase inhibitor is aSrc family tyrosine kinase inhibitor, such as but not limited to,A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271,BMS354825, CGP77675, CU201, ENMD 2076, KB SRC 4, KX2361, KX2-391, MLR1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023,XL 228, altenusin, bosutinib, damnacanthal, dasatinib, herbimycin A,indirubin, neratinib, lavendustin A, pelitinib, piceatannol,saracatinib, SrcI1, and analogs thereof. In another embodiment, thecomposition comprises more than one of the tyrosine kinase inhibitorsdisclosed herein. In yet another embodiment, the composition comprisesat least one Src family tyrosine kinase inhibitor. In such embodiments,the tyrosine kinase inhibitors may be administered together orsequentially, by different administration routes, or in the same ordifferent pharmaceutical composition.

The methods and composition disclosed herein are also useful as atreatment for a cardiovascular disease or condition in a subject havinga cardiovascular disease or condition characterized by aberrant proteintyrosine phosphorylation.

Pharmaceutical Compositions

The invention also encompasses the use of a pharmaceutical compositionof the invention to practice the methods of the invention. In oneaspect, the invention includes a pharmaceutical composition comprisingthe composition as described herein and a pharmaceutically acceptablecarrier. In another aspect, the composition described herein is used inthe manufacture of a medicament for the treatment of a cardiovasculardisease or condition in a subject in need thereof. In yet anotheraspect, the invention includes a pharmaceutical composition comprisingthe composition as described herein in combination with anothertherapeutic agent used in the treatment of a cardiovascular disease orcondition. Such pharmaceutical compositions may be provided in a formsuitable for administration to a subject, and may comprise one or morepharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The composition describedherein may comprise a physiologically acceptable salt, such as acompound contemplated within the invention in combination with aphysiologically acceptable cation or anion, as is well known in the art.

Pharmaceutical compositions that are useful in the methods of theinvention may be suitably developed for inhalational, oral, rectal,vaginal, parenteral, topical, transdermal, pulmonary, intranasal,buccal, ophthalmic, intrathecal, intravenous or another route ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations. Theroute(s) of administration will be readily apparent to the skilledartisan and will depend upon any number of factors including the typeand severity of the disease being treated, the type and age of theveterinary or human patient being treated, and the like.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

In one embodiment, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Inone embodiment, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of at least one compound ofthe invention and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers, which are useful, include, but arenot limited to, glycerol, water, saline, ethanol and otherpharmaceutically acceptable salt solutions such as phosphates and saltsof organic acids. Examples of these and other pharmaceuticallyacceptable carriers are described in Remington's Pharmaceutical Sciences(1991, Mack Publication Co., New Jersey).

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook,2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of AnimalCells” (Freshney, 2010); “Methods in Enzymology” “Handbook ofExperimental Immunology” (Weir, 1997); “Gene Transfer Vectors forMammalian Cells” (Miller and Calos, 1987); “Short Protocols in MolecularBiology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles,Applications and Troubleshooting”, (Babar, 2011); “Current Protocols inImmunology” (Coligan, 2002). These techniques are applicable to theproduction of the polynucleotides and polypeptides of the invention,and, as such, may be considered in making and practicing the invention.Particularly useful techniques for particular embodiments will bediscussed in the sections that follow.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out embodiments of the presentinvention, and are not to be construed as limiting in any way.

Noonan syndrome (NS) is an autosomal dominant disorder caused byactivating mutations in the PTPN11 gene encoding Shp2, which manifestsin congenital heart disease, short stature, and facial dysmorphia. Thecomplexity of Shp2 signaling is exemplified by the observation thatNoonans syndrome with multiple lentigines (NSML) patients possessinactivating PTPN11 mutations yet exhibit similar symptoms to NS.“Protein zero-related” (PZR), a transmembrane glycoprotein thatinterfaces with the extracellular matrix to promote cell migration, wasidentified as a major hyper-tyrosyl-phosphorylated protein in mousemodels of NS and NSML. PZR hyper-tyrosyl phosphorylation was facilitatedin a phosphatase-independent manner by enhanced Src recruitment to NSand NSML Shp2. Hence, PZR was identified as an NS and NSML target.Enhanced PZR-mediated membrane recruitment of Shp2 served as a commonmechanism to direct overlapping pathophysiological characteristics ofthese PTPN11 mutations.

The Materials and Methods used in the performance of the experimentsdisclosed herein are now described.

Antibodies, Chemicals, Cell Lines, and Expression Reagents.

Rabbit monoclonal phospho-PZR(Y241) and rabbit monoclonalphospho-PZR(Y263) antibodies were generated in collaboration with CellSignaling. Mouse monoclonal Src antibody, rabbit polyclonal Srcantibody, rabbit polyclonal phospho-ERK1/2(T202 Y204), mouse monoclonalERK1/2 antibody, rabbit polyclonal phospho-Akt(S473) antibodies andmouse monoclonal Akt antibodies were purchased from Cell Signaling.Rabbit polyclonal Shp2 antibodies and rabbit polyclonal ERK1/2 antibodywere purchased from Santa Cruz Biotechnology. Mouse monoclonal Shp2antibody was purchased from BD Bioscience. Mouse antiphosphotyrosineantibody 4G10 (05-321) was from Merck Millipore, rabbit anti-GFP (TP401)was from Acris, and mouse anti-HA.11 clone 16B12 was from Covance.Rabbit polyclonal PZR (105-6) was generously provided by Z. J. Zhao. Srcfamily kinase inhibitors PP2 and SU6656 were purchased from Calbiochem.HEK-293, NIH 3T3, SYF (Src^(−/−) Yes^(−/−) Fyn^(−/−) mouse embryonicfibroblasts [MEFs]), and Src⁺⁺ (Src-overexpressing SYF) cells werepurchased from ATCC and grown in growth medium (Dulbecco's modifiedEagle's medium [DMEM] supplemented with 1% penicillin—streptomycin and10% fetal bovine serum) in a 5% CO2 incubator at 37° C.Replication-deficient adenoviral (Ad) constructs harboring wild-typeShp2 (Ad-Shp2 WT), the E76A gain-of-function Shp2 mutant (Ad-Shp2E76A),and green fluorescent protein (GFP) (Ad-GFP) were prepared as previouslydescribed (Eminaga, S., et al., J. Biol. Chem., 283:15328-15338). NIH3T3 and SYF cells were infected with adenovirus at a dosage of 50multiplicities of infection (MOI). The pJ3Ω vectors containing SrcWT andthe K295R/Y527F dominant-negative Src mutant (SrcK295R/Y527F) have beendescribed previously (Fornaro, M., et al., J. Cell Biol., 175:87-97).The pIRES-GFP plasmids encoding the Shp2 WT, gain-of-function/Noonansyndrome mutants of Shp2 (Shp2E76A and Shp2N308D), and Noonan syndromewith multiple lentigines mutants of Shp2 (Shp2Y279C and Shp2T468M) havebeen described previously (Kontaridis, M I, et al., J. Biol. Chem.,281:6785-6792). The zebrafish Shp2 mutants have been cloned previously(Jopling, C., et al., PLoS Genet., 3:e225). The zebrafish PZR (zPZR) wascloned by nested PCR from zebrafish embryo cDNA (from bud stage to 48 hpostfertilization [hpf]). The zPZR ITIM Y236F, Y258F, and Y236F Y258Fmutants were made using site-directed mutagenesis. RPTPa signal sequenceand Hemagglutinin (HA) tag were incorporated into N-terminus of zPZR.DNA transfection into HEK-293 and SYF cells was performed usingLipofectamine 2000 according to the manufacturer's protocol.

MS Analysis.

The PhosphoScan method was performed as previously described (Rikova,K., et al., Cell, 131:1190-1203). Wild-type and Shp2 mutant (Noonansyndrome) mouse hearts were homogenized, sonicated, and centrifuged toremove cellular debris. Total protein for each tissue was normalizedusing the ProteinPlus Coomassie reagent (Pierce), and proteins werereduced, alkylated, and digested overnight using trypsin (Worthington).The resulting peptides were separated from non-peptide material bysolid-phase extraction with Sep-Pak classic C₁₈ cartridges (Waters).Lyophilized peptides were redissolved, and phosphopeptides were enrichedby immunoaffinity purification using pY-100 phosphotyrosine antibody(9411; Cell Signaling Signaling Technology). Peptides were eluted with0.15% trifluoroacetic acid (TFA) and concentrated with C₁₈ spin tipsimmediately prior to liquid chromatography-mass spectrometry (LC-MS)analysis. Duplicate injections of each sample were run to generateanalytical replicates and increase the number of tandem MS (MS/MS)identifications from each sample. Peptides were loaded directly onto a10-cm by 75-μm PicoFrit capillary column packed with Magic C₁₈ AQreversed phase resin. The column was developed with a 45-min lineargradient of acetonitrile in 0.125% formic acid delivered at 280 nl/min.Tandem mass spectra were collected with an LTQ-Orbitrap XL massspectrometer running XCalibur using a Top 10 method, a dynamic exclusionrepeat count of 1, and a repeat duration of 30 s. MS spectra werecollected in the Orbitrap component of the mass spectrometer, and MS/MSspectra were collected in the LTQ portion. MS/MS spectra were processedusing SEQUEST and the Core platform (Gygi Lab, Harvard University).Searches were performed against the mouse NCBI database, with reversedecoy databases included for all searches to estimate false-positiverates. Peptide assignments were obtained using a 0.98-precision cutoffin the linear discriminant analysis module of Core. Cysteinecarboxamidomethylation was specified as a static modification, andmethionine oxidation and serine, threonine, and tyrosine phosphorylationwere allowed. Results were further narrowed using mass accuracy (5-ppm)filters and the presence of a phosphotyrosine in the peptide. Label-freequantitation was performed using Progenesis v4.1 (Nonlinear Dynamics).Peptide abundance data were manually reviewed in Progenesis for allpeptides with at least a 2.0-fold change to ensure accuracy of results.

Animal Handling—Ptpn11^(D61G/+) mice were provided from Dr. BenjaminNeel (University of Toronto, Toronto) and were genotyped as describedpreviously (Araki T, Mohi M G, Ismat F A, Bronson R T, Williams I R,Kutok J L, Yang W, Pao L I, Gilliland D G, Epstein J A, Neel B G. 2004.Mouse model of Noonan syndrome reveals cell type- and genedosage-dependent effects of Ptpn11 mutation. Nat Med 10:849-857).Briefly, Ptpn11^(D61G/+) male mice were crossed with WI C75BL/6×SV12.9female mice and their offspring were genotyped by PCR and digestion withAga for D61G allele.

Dasatinib Treatment—

N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide monohydrate (dasatinib, BMS-354825) was purchased fromBiovision. dasatinib was dissolved in DMSO at a concentration of 10mg/ml, then resuspended in vehicle (1× Dulbecco's PBS) at aconcentration of 200 μg/ml. WT and Ptpn11^(D61G/+) male mice wereinjected daily with dasatinib (0.1 mg/kg, i.p.), beginning at postnatalday 10 until 6-weeks after birth (P42). And then, injection wascontinued or discontinued for 2 weeks, Vehicle-injected mice served ascontrol, Body weight was measured weekly and echocardiography wasperformed at P42 (6-weeks) and P56 (8-weeks). Animal handling wasapproved by The Yale University Institutional Animal Care and UseCommittee.

Echocardiographic Studies—

Cardiac dimensions and function were analyzed by echocardiography usinga Vevo 770 console, Mice were lightly anesthetized with inhaledisoflurane (0.2% in O₂). All measurements were obtained from three tosix consecutive cardiac cycles, and the averaged values used foranalysis. Interventricular septum wall (IVS), left ventricular internaldimension (LVID) and left ventricular posterior wall thickness (LVPW) inboth end-diastolic (d), end-systolic (s) were measured from the andM-mode tracings. Diastolic measurements were performed using theleading-edge method of the American Society of Echocardiography. ForTM-mode measurements, left ventricular end-diastolic volume (LV vol,d)and end-systolic volume (LV vol,s) were calculated. Ejection fractionpercentage (EF) was calculated as [(LVvol,d−LVvol,s)/VLvol,d]×100, andfractional shortening percentage was calculated as[(LVID,d−LVID,s)/LVID,d]×100.

Statistical Analysis—

Statistical values are presented as the mean±s.e.m. A two-way ANOVA(Tukey's multiple comparisons) test was used to calculate the P values.All statistical analyses were performed with GraphPad Prism 5. For allstudies, a P value less than 0.05 was considered significant.

The Materials and Methods used in the performance of the experiments inExample 2 disclosed herein are now described.

Antibodies, Chemicals, Cell Lines and Plasmids—

The following antibodies were used either for immunoblotting (IB) orimmunoprecipitation (IP) as indicated. Mouse monoclonal Flag (F1804,IP-1:100, IB-1:1,000) and mouse monoclonal biotinylated Flag (F9291,IB-1:1,000) antibodies were from Sigma. Mouse monoclonal Myc (sc-40,IP-1:100, IB-1:1,000), mouse monoclonal biotinylated Myc (sc-40B,IB-1:1,000), rabbit polyclonal Shp2 (sc-280, IB-1:1,000), mousemonoclonal p38 (sc-535, IB-1:1,000), mouse monoclonal GST (sc-138,IB-1:1,000) antibodies were from Santa Cruz Biotechnology. Rabbitmonoclonal phospho-PZR (Y241; #8181, IB-1:1,000), rabbit monoclonalphospho-PZR (Y263; #8088, IB-1:1,000), rabbit polyclonal phospho-Src(Y416; #2101, IB-1:1,000), mouse monoclonal Src (#2110, IB-1:1,000),mouse monoclonal Raf1 (#12552, IB-1:1,000), rabbit polyclonalphospho-MEK1/2 (S217/221; #9154, IB-1:1,000) mouse monoclonal MEK1/2(#4694, IB-1:1,000), rabbit polyclonal phospho-ERK1/2 (T202/Y204; #9101,IB-1:1,000), mouse monoclonal ERK (#9107, IB-1:1,000), rabbit polyclonalphospho-p38(T180/Y182; #9215, IB-1:1,000), rabbit polyclonal phospho-JNK(T183/Y185; #4668, IB-1:1,000), mouse monoclonal JNK (#3708,IB-1:1,000), rabbit polyclonal phospho-Akt(5473; #9271, IB-1:1,000),mouse monoclonal Akt (#2967, IB-1:1,000), rabbit polyclonal SERCA2A(#9580, IB-1:1,000), rabbit polyclonal Troponin I (#4002, IB-1:1,000)antibodies were purchased from Cell Signaling. Rabbit polyclonalphospho-Raf1(Y341; ab192820, IB-1:1,000) and rabbit polyclonal alphatubulin (ab4074, IB-1:1,000) antibodies were obtained from Abcam. Mousemonoclonal Shp2 (#610622, IB-1:1,000) antibody was purchased from BDBiosciences. Mouse monoclonal His (#11922416, IB-1:1,000) antibody wasfrom Roche. Rabbit polyclonal Troponin T (MS-295, IB-1:1,000) was fromThermo Scientific. Rabbit polyclonal PZR antibody (IB-1:1,000) wasgenerously provided by Z. J. Zhao. Dasatinib was purchased fromBiovision and STI-571 was obtained from LKT laboratories. Shp2phosphatase inhibitor was generously provided by Z.-Y. Zhang (IndianaUniversity). HEK-293T cells were purchased from ATCC and mouse embryonicfibroblasts (MEFs) were isolated from WT and Ptpn11^(D61G/+) mice. Cellswere grown in growth medium (Dulbecco's modified Eagle's medium [DMEM]supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum)in a 5% CO₂ incubator at 37° C. Human Src and Ptpn11 full length, N+Cand PTP constructs were generated by PCR and cloned into the pCMV-3Tag4aand pCMV-Tag2b (Clontech laboratories) vectors. DNA transfection intoHEK-293T cells was performed using Lipofectamine 3000 (Invitrogen)according to the manufacturer's protocol.

Immunoprecipitation and Immunoblotting—

Cells or heart tissue were lysed on ice in lysis buffer (25 mM Tris-HO,pH 7.4, 136 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 1% Nonidet P-40, 1 mMNa₃VO₄, 10 mM NaF, 1 mM benzamidine, 1 mM PMSF, 1 μg/ml pepstatin A, 5μg/ml aprotinin, and 5 μg/ml leupeptin). Cell or tissue lysates wereincubated at 4° C. for 30 min and clarified by centrifugation at 14,000rpm at 4° C. for 10 min. Protein concentration was determined using theBCA reagent according to the manufacturer's instructions (Pierce). Forimmunoprecipitations, 500 μg of lysate was incubated with 1 μg ofindicated antibodies at 4° C. for overnight. Immune complexes werecollected on either protein A- or protein G-Sepharose beads for 4 hr at4° C., washed three times with same lysis buffer and then heated to 95°C. in sample buffer for 5 min. Total lysates or immune complexes weresubjected to SDS-PAGE and immunoblotting. The sites of antibody bindingwere visualized using enhanced chemiluminescence detection or OdysseyImaging System.

In Vitro GST-Pull Down Assay—

Bacterial purified GST-SH3 of Src and His-PTP of Shp2 were provided byT. Boggon (Yale University). Pull-down assays were carried out in 1 mllysis buffer containing GST-SH3 of Src protein with either His-PTP ofShp2 or Flag-tagged Shp2 overexpressing HEK-293 cell lysates forovernight at 4° C. SH3-bound Shp2 proteins were affinity purified byBSA-coated GST-Sepharose beads for 1 hr at 4° C. The interaction betweenSH3 of Src and Shp2 proteins was examined using immunoblotting withanti-His or anti-Flag and anti-GST antibodies.

Animal Handling—

Ptpn11^(D61G/+) mice were provided from Dr. Benjamin Neel (University ofToronto, Toronto) and were genotyped as described previously⁹. Briefly,Ptpn11^(D61G/+) male mice were crossed with wild type C75BL/6×SV 129female mice and their offspring were genotyped by PCR and digestion withAgeI for the D61G allele. Dasatinib (Biovision) was suspended in vehicle(1% DMSO in phosphate buffer saline), For prenatal treatment, dasatinibwas injected i.p. (0.1 mg/kg body weight) into pregnant mice daily,beginning on gestational day 7.5 (E7.5) continuing (in nursing females)until postnatal day 9. Vehicle-injected mice served as control.Beginning at P10, dasatinib or vehicle alone was injected (i.p.)directly into pups daily, until 8-weeks after birth. For postnataltreatment, dasatinib was injected (i.p.) into pups at P10, until 6-weeksafter birth; injection was discontinued for 2-weeks. Animal handling wasapproved by The Yale University Institutional Animal Care and UseCommittee.

Histology—

Heart, liver and spleen were isolated from vehicle- or dasatinib-treatedwild type and NS mice. Tissues were fixed in 4% paraformaldehyde inphosphate-buffer saline (PBS), processed for paraffin sections andstained with hematoxylin and eosin (H&E) or Masson's Trichrome. Tissueimages were obtained under bright field microscopy (Olympus BX51, YaleLiver Center).

Echocardiography—

Mice were anesthetized in a sealed plastic chamber with 1% isofurane inoxygen until immobile, and then were transferred onto a heated procedureboard (37° C.). Animal were kept anesthetized with 1% isofluranesupplied by a nose cone connected to the isoflurane vaporizer during theentire procedure. The scan head was placed on the chest of the mouse andstable image signals (both B mode and M mode) were acquired and dataanalyzed with Vevo 770 (VisualSonics). Systolic and distolic leftventricle peripheral wall thickness, chamber diameter andinterventricular wall thickness were measured with M mode image.Percentage of ejection fraction (EF) and fractional shortening (FS) werecalculated.

Hemodynamic Study—

Anesthesia was induced by intraperitoneal injection of ketamine (100mg/kg) and xylazine (5 mg/kg). The animal was placed on a warm pad andan incision was made on the neck. The right side carotid artery wasexposed and a 1.9-French transducer-tipped catheter (Millar Inc.,Houston, Tex.) was inserted into the artery, and then was advanced intoleft ventricle. Left ventricular pressures including high-fidelitypositive, negative dp/dt and heart rate were measured under basalconditions. Data were recorded and analyzed by using LabChart software.

RNA Extraction and Quantitative Real-Time PCR Analysis—

RNA was isolated from mice heart using an RNeasy kit (Qiagen, CA)according to the manufacturer's instructions. A total of 1 μg RNA wasreverse transcribed to generate cDNA using a reverse transcriptase PCRkit (Applied Biosystems, CA). Real-time quantitative PCR was carried outin triplicate using the Applied Biosystems 7500 Fast real-time PCRsystem and SYBR green gene expression master mix with following primerpairs.

18S rRNA, SEQ ID NO: 1 5′-ACCGCAGCTAGGAATAATGGA-3′, SEQ ID NO: 25′-ACCAAAAGCCTTGACTCCG-3′; ANF, SEQ ID NO: 35′-CCTGGAGGAGAAGATGCCGGTAGAA-3′, SEQ ID NO: 45′-CCCCAGTCCAGGGAGGCACCTCGG-3′; BNP, SEQ ID NO: 55′-CACTTCAAAGGTGGTCCCAGAGCTGC-3′, SEQ ID NO: 65′-GACCGGATCGGATCCGTCAGTCG-3′; αMHC, SEQ ID NO: 75′-GTCCCGGACACTGGACCAGGCC-3′, SEQ ID NO: 85′-CTCCTTTTCTTCCAGTTGCCTAGCCAA-3′; βMHC, SEQ ID NO: 95′-GAGCAAGGCCGAGGAGACGCAGCGT-3′, SEQ ID NO: 105′-GAGCCTCCTTCTCGTCCAGCTGCCGG-3′; Col1a2, SEQ ID NO: 115′-AGGTCTTCCTGGAGCTGATG-3′, SEQ ID NO: 12 5′-ACCCACAGGGCCTTCTTTAC-3′;Col3a1, SEQ ID NO: 13 5′-ACAGCAAATTCACTTACACAGTTC-3′, SEQ ID NO: 145′-CTCATTGCCTTGCGTGTTT-3′.

All relative gene expression levels were analyzed using the ΔC_(T)method and normalized to 18S rRNA expression.

Enzymatic Digestion of Cardiac Tissue for Single-Cell Analysis—

Cardiomyocytes from 8 week old mice were isolated by a Langendorffprocedure, modified from Xianghua Xu, et al. J Vis Exp. 2009; (28):1308.In brief, the hearts were quickly excised and cannulated to aLangendorff apparatus where they were perfused with 37° C. Ca²⁺-freeperfusion buffer (in 25 mM HEPES, 118 mM NaCl, 4.8 mM KCl, 2.0 mMKH₂PO₄, 2.55 mM MgSO₄, 10 mM BDM and 10 mM Glucose). To digest thetissue, the heart was perfused with buffer containing 0.5 mg/mL LiberaseTH (Roche Applied Science, Penzberg, Germany). After ˜10 min, the heartwas removed from the Langendorff apparatus and the right ventricle andatria removed. The left ventricle was isolated, cut into small piecesand digested further at 37° C. digestion solution with mechanicalagitation for 5-10 min, and then gently triturated to liberateindividual cells. The remaining tissue chunks were transferred to freshdigestion buffer and the process was repeated up to 6 times, or untilall tissue was digested. Cells were removed from collagenase by gentlecentrifugation and resuspended in several washing steps of buffercontaining FBS and gradually reintroduced to calcium (0.05-1.1 mM)through step-wise additions of concentrated CaCl₂ solution. Cells wereallowed to rest for at least 1 h before imaging.

Cardiomyocyte Functional Characterization—

Cardiomyocytes were imaged in Tyrodes solution (in 150 mM: NaCl: 140,KCl: 5.4, CaCl₂:1.8, MgCl₂:1, HEPES: 25 mM, glucose: 10 mM). Cellpellets were loaded for 15 minutes in the dark with Tyrodes supplementedwith 2.5 μM Fura-2 AM complimented with pluronic acid (20% w/v), forcalcium fluorescence imaging. After 15 minutes of loading, the cellswere resuspended in fresh Tyrodes solution and allowed to settle untilimaging. Cardiomyocyte Ca²⁺ transients and unloaded shorteningcontractions were measured using an inverted microscope (Nikon Eclipse,Chiyoda, Tokyo) equipped with a temperature controlled perfusion bath(Cell MicroControls, Norfolk, Va.) under constant perfusion of 37° C.Tyrodes solution. Cells were field-stimulated at 1 Hz. Contractileevents were imaged in real-time using a sarcomere length camera system(HVSL, Aurora Scientific, Ontario, Canada). Only rod-shaped cells withwell-defined sarcomere striations that contracted when stimulated wereselected for measurement. Sarcomere length was measured and recorded forten consecutive beats and subsequently averaged across beats to producea single waveform. Calcium transient measurements were recordedsimultaneously using alternating excitation wavelengths of 340 and 380nm generated at an overall rate of 100 Hz by a RatioMaster fluorescencesystem (PTI, Birmingham, N.J.). Fluorescence emission was filtered at acenter wavelength of 510 nm and quantified to obtain responses to thealternating excitation wavelengths (F₃₄₀ and F₃₈₀, respectively). Ca²⁺transients were reported as the interpolated ratio of the twofluorescence intensities (F₃₄₀/F₃₈₀) at each time point. Data wererecorded using a DAP5216a data acquisition system (MicrostarLaboratories, Bellevue, Wash.) and processed using custom softwarewritten in MATLAB (MathWorks, Natick, Mass.). Peak sarcomere lengthshortening (Peak SL shortening), time to peak shortening (TTP), time to50% re-lengthening (RT50), the magnitude of the calcium transient (Ca²⁺R_(mag): Max F₃₄₀/F₃₈₀−Min F₃₄₀/F₃₈₀) and the rate of calcium decay, Tau(Tau_(Ca2+)) were computed.

Statistical Analysis—

All data represent the means±standard errors of the means (SEM).Differences between groups were assessed using analysis of variance(ANOVA) with Tukey multiple comparisons using the GraphPad Prism 6statistical software program.

The Results of the experiments disclosed herein are now described.

Example 1: Targeting RASopathy-Mediated Cardiac Disease with TyrosineKinase Intervention

FIG. 1A is an illustration of a proteomic analysis of differentiallytyrosyl-phosphorylated proteins in hearts of Ptpn11^(D61G/+) mice.

FIG. 1B is graph showing log 2-transformed values for the ratio of eachphosphotyrosine-containing peptide in wild-type and Ptpn11^(D61G/+)mouse hearts. FIG. 1C is a heat map of differentiallytyrosyl-phosphorylated peptides (the site of phosphorylation isidentified by MS in parentheses). FIG. 1D is a panel of images ofextracted ion chromatogram and peptide sequence of PZR-containingtyrosine 242 (upper panels) and tyrosine 264 (lower panels) bydifferential proteomics. FIG. 1E shows amino acid sequences of the PZR Cterminus in different vertebrates. Global phosphotyrosyl proteomics inthe hearts of mice harboring a knockin mutation of the Noonan syndromemutant (Shp2^(D61G/+)) reveals altered regulation of tyrosylphosphorylated proteins. MS analysis shows that the most abundantlyhypertyrosyl phosphorylated protein is PZR in these mice. Tyrosylresidues 264 and 242 were identified to be the sites of PZR tyrosylphosphorylation that were increased in the hearts of Noonan syndrome(Shp2^(D61G/+)) mice. PZR tyrosine 242 and 264 are likely to beimportant for the function of PZR given that they are highly conservedthroughout evolution. These results suggest that increased PZR tyrosylphosphorylation may play a role in the pathogenesis of Noonansyndrome-related cardiac disease. These results identified Y242 and Y264as sites of PZR hypertyrosyl phosphorylation in this mouse model of NS.

FIG. 2A-2E show immunoblots showing the characterization of PZR tyrosylphosphorylation. Conformation of the site of PZR hyper tyrosylphosphorylation by NS and NSML mutants. Expression of aphosphorylation-resistant mutant of PZR at the site(s) of tyrosylphosphorylation found in mice impairs its ability to be phosphorylatedin cultured cells also as detected using phospho-specific anti-PZR(Y242)and anti-PZR(Y263). Mutants of Shp2 that represent those found either inNS or NSML patients are capable of inducing PZR hypertyrosylphosphorylation at Y241 and Y263. Similarly, zebrafish PZR exhibitsidentical properties of being able to be tyrosyl phosphorylated at thecomparable residue. These results suggested NS or Leopardsyndrome-associated Shp2 mutants induced PZR hyperphosphorylation invarious cell lines.

FIGS. 3A-3D show PZR tyrosyl phosphorylation in the heart and the cortexof Ptpn11^(D61G/+) and Ptpn11^(Y279C/+) mice. Using site-specificphospho-PZR antibodies we show that mice expressing a knockin mutationof the Shp2^(D61G/+) allele exhibit increased PZR tyrosylphosphorylation in the heart and cortex. Similarly, mice expressing aknockin mutation of the Shp2^(Y279C/+) allele exhibit increased PZRtyrosyl phosphorylation in the heart and cortex. These resultsdemonstrate that both NS and NSML mutations, with enhanced and reducedphosphatase catalytic activity, respectively are capable of increasingPZR tyrosyl phosphorylation. These results demonstrate that PZR is atarget for both NS and NSML and suggest that PZR represents a novelcommon signaling component of these RASopthies. The results showed thatPZR hypertyrosyl phosphorylation in the heart and cortex of NS and NSMLmodel mice. These in vivo data confirmed the in silico (FIGS. 1A-1E) andin vitro (FIGS. 2A-2E) experiments

FIGS. 4A-4C show images and graphs of PZR tyrosyl phosphorylation inPtpn11^(D61G/+) mice liver, kidney and spleen. PZR is hypertyrosylphosphorylated in the liver, kidney and spleen of Ptpn11^(D61G/+) mice.These results showed PZR hypertyrosyl phosphorylation in the varioustissues of NS mice.

FIGS. 5A-5D show images of ERK and Akt phosphorylation in the heart andthe cortex of Ptpn11^(D61G/+) and Ptpn11^(Y279C/+) mice. Phosphorylationstatus of ERK and AKT in the heart and cortex of Ptpn11^(D61G/+) miceindicates no substantive differences to that of wild type despite undersimilar conditions PZR is hypertyrosyl phosphorylated (see FIGS. 3A-3D).These results implicated no apparent differences were observed in thebasal levels of Shp2, phospho-ERK1/2, or phospho-Akt between either NSor NSML mice. These results further indicated that the effects of theseRASopathies on MAPK and AKT signaling in the heart and cortex weredistinct from those that drove PZR hyper tyrosyl phosphorylation.

FIGS. 6A-6B are blots showing the effect of Src family kinases onNS/NMLS-Shp2 mediated PZR Y241 and Y263 phosphorylation. NS andNMLS-associated mutants induce PZR hypertyrosyl which can be inhibitedupon pre-treatment of cells with the SFK inhibitor SU6656. These resultssuggested that the SFK's were capable of phosphorylating PZR on bothY241 and Y263. These results further showed that NS and NSML-Shp2mutatns induced PZR hypertyrosyl phosphorylation is Src family kinasedependent.

FIGS. 7A-7B are blots showing that Src kinase mediated NS/LS-Shp2induced PZR hyper-tyrosyl phosphorylation. FIGS. 7C-7D are blots showingthat Src kinase mediated PZR hyper-tyrosyl phosphorylation. The figuresshow the comparison of the effects of tyrosine kinase inhibitory potencyon PZR tyrosyl phosphorylation. The tyrosine kinase inhibitors, PP2 andSU6656, were administered to cells expressing an activated Shp2(Shp2-E76A) mutant. Although both PP2 and SU6656 were capable ofinhibiting Shp2-E76A-induced PZR hypertyrosyl phosphorylation, SU6656was more effective. PZR hyper tyrosyl phosphorylation was inhibitedcompletely at 1 μM SU6656 as compared with PP2, which inhibited at 5 μM.These results supported the notion that the Src family kinases wereresponsible for the phosphorylation of PZR. Importantly, thephosphorylation of PZR by Src created a binding site (pY241/pY263) forShp2 to interact with PZR. These results implicated c-Src as directlyphosphorylating PZR at Shp2 binding sites.

In Noonan syndrome, the increased phosphorylation of Y241 and Y263 bySrc resulted in deleteriously high levels of PZR/Shp2 complex which isproposed to be a mechanism that drives the development of congenitalheart disease in these patients.

FIGS. 8A-8B show enhanced Src complex formation with NS/NSML-associatedShp2 mutants and PZR. FIG. 8A shows that mutant forms of Shp2 that areknown to cause either NS or NSML can bind with increased affinity toc-Src as compared with wild type Shp2. These results further suggestthat c-Src directly phosphorylated PZR at Shp2 binding sites.

FIG. 9 is an illustration of the model for the effects of NS- andNSML-Shp2 mutants on PZR tyrosyl phosphorylation. The model is basedupon experimental data from which PZR was observed as hypertyrosylphosphorylated on Shp2 binding sites in the heart of both NS and NSMLmouse models. The increased PZR tyrosyl phosphorylation promotedincreased Shp2 binding to PZR. The diagram proposes that enhanced PZRtyrosyl phosphorylation results in increase recruitment of Shp2 to PZRto promote further PZR tyrosyl phosphorylation, as well as otherpotential Src substrates that are in close proximity.

In addition, NS and NSML mutants interacted with the tyrosine kinase,Src, with increased affinity. Together, these promiscuous interactionsresulted in dysfunctional downstream signaling from PZR whichcontributed to the development of congenital heart disease. It isproposed that by intervening with Src tyrosine kinase activity, PZR/Shp2complexes are reduced and altered signaling from PZR, and possibly othertargets, are corrected.

FIG. 10 is a panel of images showing dosing of dasatinib. MalePtpn11D61G/+ mice were injected intrapertinoeally with dasatinib at theindicated dose or DMSO control. Twenty-four hours later, the mice weresacrificed and heart tissues were harvested and immunoblotted usingtotal PZR and pY(263)-PZR antibodies. These results showed thatinjection of dasatinib in NS mice was effective at reducing PZR tyrosylphosphorylation.

FIG. 11A is an illustration showing the pre-natal dosing regimen fordasatinib in NS mouse model. Dasatinib was administered daily topregnant mothers between the time of when animals were in utero at E7.5until 9 days after birth (P9). At 10 days after birth (P10), NS micereceived dasatinib directly by daily injections (i.p.) for 6 weeks (P42)and 8 weeks (P56).

FIG. 11B is an illustration showing the post-natal dosing regimen fordasatinib in NS mouse model. Dasatinib was administered to NS micestarting at 10 days after birth (P10) daily for 6 weeks (P42) after 6weeks treatment was stopped and heart function measured. This same groupof mice was then evaluated after 2 weeks of being withdrawn fromdasatinib treatment.

In FIGS. 11A and 11B, the mice were evaluated for cardiac function at 6and 8 weeks. These illustrations described the pre- or post-natalDasatinb treatment strategy into NS mice. The dosing regimens describedherein were designed to test three aspects of the effectiveness ofdasatinib for therapeutic intervention of NS-related cardiac disease.Because NS is a developmental disorder, the first dosing regimen shownin FIG. 11A tested the effectiveness of dasatinib in exerting atherapeutic effect when administered to the developing embryo. Thesecond evaluation determined the effectiveness of dasatinib in treatingNS-related cardiac disease when administered after birth. This strategywas linked more closely with cardiac disease outcomes as it has beenrealized that patients could be administered therapeutic doses ofdasatinib after birth. This dosing strategy would mitigate risk of inutero complications. Finally, the third test was to establish that upontherapeutic cardiac function improvement, dasatinib administration wasnecessary to maintain treatment.

FIG. 12A is a panel of graphs showing dasatinib-treated pre-natally inNS mice improves cardiac function (P42, seen in FIG. 11A). FIG. 12B is apanel of graphs showing dasatinib-treated in post-natal NS mice improvescardiac function (P42). FIG. 12C is a panel of graphs showing preservedimprovement of cardiac function after cessation of dasatinib treatment(P56, seen in FIG. 11B). These results provided evidence for theinvolvement of Src signaling in the pathogenesis of NS.

The results of these experiments demonstrated that a low dose ofdasatinib—which is defined here as a dose that is below that found to beefficacious for the treatment of cancer—is effective in improvingcardiac function in NS mice. In FIG. 12A, the results demonstrated thatheart function, as measured by the ejection fraction (EF) and thefractional shortening (FS), were completely restored to wild typeparameters when dasatinib was injected into pregnant mice. In FIG. 11B,dasatinib was shown to be effective even when administeredpost-developmentally. The treatment still exerted complete correctivefunctionality in cardiac function in NS mice. These results indicatedthat therapeutic administration of dasatinib for the treatment ofcongenital heart disease in RASopathy patients can occur withsubstantially less risk by post-development administration. Finally,FIG. 11C shows that cessation of dasatinib for 2 weeks after effectivecardiac functionality has been achieved resulted in continuedpreservation of cardiac function. These results demonstrated that onceeffective therapy has been attained and heart functionality hasreturned, the need for continued exposure to dasatinib was unnecessary.

Example 2: Selective Rescue of Cardiac Defects in a Mouse Model ofNoonan Syndrome (NS) by Dasatinib

Shp2 is comprised of two Src homology 2 (SH2) domains, a proteintyrosine phosphatase (PTP) domain and a carboxy-terminal tail.NS-associated Shp2 (NS-Shp2) mutations often occur in amino acidresidues that occupy the interface between the amino terminal SH2 andPTP domains. The resultant mutations disrupt the auto-inhibitory“closed” conformation that occurs between the SH2 and PTP domain, infavor of a more “open” configuration that facilitates catalysis.

The protein zero-related protein (PZR), a transmembrane glycoproteinwhich contains two immunoreceptor tyrosine-based inhibitory motifs(ITIMs) in its C-terminus, serves as a c-Src substrate and constitutes amajor hyper-tyrosyl phosphorylated protein and Shp2 binding target inthe heart of a mouse model of NS. NS-Shp2 mutants interact with enhancedaffinity to c-Src endowing these mutants with the ability topromiscuously target c-Src through PZR complex formation. Usingzebrafish as a model of NS-mediated CHD, it was proposed thatPZR-Shp2-Src complex formation drives aberrant signaling in NS-mediatedCHD.

Critical to this supposition is the ability of NS-Shp2 mutants toexhibit enhanced interactions with c-Src leading to increasedc-Src-mediated signaling. The nature of the enhanced interaction betweenShp2 and c-Src likely occurs as a result of increased exposure ofbinding surfaces within the PTP domain of Shp2 that are otherwiseunexposed in the “closed” conformation. Although Shp2 has been shown toform a complex with c-Src through its SH3 domain, the region on Shp2that c-Src interacts with is not yet defined.

To address this, a series of Shp2 deletion mutants were designed (FIG.13a ), co-transfected into HEK-293T cells, and complex formation wasexamined by co-immunoprecipitation (FIG. 23). As expected, full-lengthShp2 was detected in a complex with c-Src, whereas a deletion mutant ofShp2 lacking the PTP domain failed to interact (FIG. 13b ). In vitrobinding assays further established that the PTP domain of Shp2 and theSH3 domain of c-Src interacted directly (FIG. 13c ).

Since this interaction occurs within the PTP domain of Shp2, the “open”conformation of NS-Shp2 mutants are thought to be poised to establishmore stable interactions with the SH3 domain of c-Src as compared withwild type Shp2. As such, NS-Shp2 mutants complex more stably with c-Srcat the membrane through PZR, which has been proposed previously to be aputative mechanism of aberrant c-Src-mediated signaling. Importantly,these observations implicate c-Src, or a Src family kinase (SFK) member,as a candidate(s) in Shp2-mediated NS pathogenesis.

To test whether the SFKs are involved in NS pathogenesis, c-Src wasinhibited pharmacologically in order to test whether its inhibitionameliorates Shp2-NS signaling. To inhibit c-Src dasatinib (SPRYCEL©), adual Abl-Src kinase inhibitor, approved for the treatment of chronicmyeloid leukemia, was used. Treatment of mouse embryonic fibroblasts(MEFs) isolated from NS mice with dasatinib blocked c-Src, ERK1/2 andPZR tyrosyl phosphorylation (FIGS. 13d-13h and 24).

Inhibition of PZR tyrosyl phosphorylation by dasatinib also resulted indisruption of PZR/Shp2 complex formation (FIG. 13d ). Additionally,Raf-1, MEK1, JNK and Akt were inhibited in NS-derived MEFs by dasatinib(FIG. 17).

The BCR-Abl kinase inhibitor, STI-571 (Gleevec©), was ineffective atimpairing PZR tyrosyl phosphorylation, indicating that inhibition of PZRtyrosyl phosphorylation and disruption of the PZR/Shp2 complex waslikely a result of dasatinib's effect on c-Src rather than Abl (FIG.17). Moreover, an Shp2 inhibitor did not block NS-Shp2-mediated PZRhypertyrosyl phosphorylation (FIG. 17), indicating that NS-Shp2-mediatedc-Src PZR tyrosyl phosphorylation occurred independently of Shp2'sphosphatase activity.

To examine the effects of dasatinib on NS-mediated c-Src and PZR tyrosylphosphorylation in vivo, dasatinib was injected into mice containing aknockin mutation of Shp2 at Asp61 to Gly61 (D61G) (Araki et al Nat Med10, 849-857 (2004)). Mice heterozygous for PtpN11^(D61G/+), referred toherein as “NS mice,” recapitulate many features of the human disease,including short stature, craniofacial abnormalities, myeloproliferativedisease and CHD.

Dasatinib has been shown to be effective in preventing tumor incidencein mice at a dosage of ˜20 mg/kg (Shah et al., Science 305,399-401(2004)). The therapeutic effects of dasatinib in humans isreported to be ˜2 mg/kg, an equivalent dose of ˜24 mg/kg, inmice(Kantarjian et al., N Engl J Med 362, 2260-2270 (2010), Yu et al.,Clinical Cancer Research 15, 7421-7428 (2009), Apperley J Clin Oncol 27,3472-3479 (2009)). Doses of dasatinib as low as 0.5 mg/kg weresufficient to significantly inhibit both c-Src and PZR tyrosylphosphorylation in the heart of 3-week-old NS mice (FIGS. 13i-13l ).Notably, at these doses of dasatinib (0.1-0.5 mg/kg), neither ERK1/2phosphorylation (FIGS. 13i-13l ), Raf-1, MEK1, p38 MAPK, nor JNK wereaffected in the heart of 3-week-old NS mice (FIG. 18). These resultsindicate that dasatinib, at up to 250-fold lower doses than theeffective chemotherapeutic doses (for chronic myelogous leukemia adultpatient approx. 100-140 mg/day or approx. 1.4-2.0 mg/kg/day), caninhibit PZR tyrosyl phosphorylation in the heart of NS mice.

Moreover, in the hearts of NS mice, dasatinib-mediated inhibition of PZRtyrosyl phosphorylation was uncoupled from the inhibition of ERK1/2phosphorylation (FIG. 13). Hence, low doses of dasatinib interfered withNS-Shp2 signaling independently of the ERK1/2 pathway.

Dasatinib was administered into pregnant mothers and amelioration ofcardiac defects in NS mice was assessed. Dasatinib was administered at0.1, 0.5 or 1.0 mg/kg daily interperitoneally into wild type pregnantmice intercrossed with NS mice, beginning at embryonic day 7 untilpostnatal day 9 (in nursing females). After postnatal day 10 (P10),dasatinib injections resumed directly into individual pups daily until8-weeks (P56) after birth (FIG. 14a ). Although dasatinib treatment at0.5 and 1.0 mg/kg/per day showed embryonic lethality, dasatinibtreatment at 0.1 mg/kg/per day had no observable adverse effects (Table1).

Cardiac function of NS mice was examined at 6- and 8-weeks byechocardiography and invasive hemodynamics. The ejection fraction (EF)and fractional shortening (FS) in untreated NS mice was significantlyreduced by 35% (P<0.01) as compared with vehicle-treated wild type mice.However, dasatinib-treated NS mice at P42 exhibited complete restorationof cardiac function as compared with vehicle-treated NS mice (FIGS. 14band 14c and Table 2). However, continued administration of dasatinib foran additional 2 weeks induced cardiac failure in both wild type anddasatinib-treated NS mice (FIGS. 14d-14e and Table 3). These datasuggest that dasatinib treatment in utero can rescue the impairedcardiac function observed in NS mice. Thus, c-Src activity contributesto the manifestation of Shp2-NS CHD.

TABLE 1 Progeny from prenatal dasatinib treated Ptpn11^(D61G/+) X WTbreeder. Ptpn11^(+/+) Ptpn11^(D61G/+) Dead pups Vehicle 21 20 Dasatinib(0.1 mg/kg) 25 19 4

TABLE 2 Echocardiography parameters of prenatal vehicle- ordasatinib-treated WT and Ptpn11D^(61G/+) mice at P42. Vehicle DasatinibWT (n = 8) D61G/+ (n = 8) WT (n = 8) D61G/+ (n = 8) IVS,d (mm) 0.69 ±0.06  0.57 ± 0.03 0.67 ± 0.04  0.65 ± 0.06 IVS,s (mm) 1.10 ± 0.06  1.01± 0.06 1.08 ± 0.09  0.97 ± 0.10 LVID,d (mm) 3.95 ± 0.15  3.55 ± 0.103.93 ± 0.08  3.61 ± 0.12 LVID,s (mm) 2.45 ± 0.19  3.01 ± 0.07** 2.49 ±0.09  2.32 ± 0.12^(††) LVPW,d (mm) 0.74 ± 0.03  0.78 ± 0.02 0.76 ± 0.05 0.70 ± 0.04 LVPW,s (mm) 1.23 ± 0.08  0.97 ± 0.06* 1.13 ± 0.04  1.12 ±0.07 LV vol,d (mm³) 68.02 ± 4.23  55.82 ± 2.96 67.51 ± 2.54  60.93 ±3.75 LV vol,s (mm³) 20.15 ± 3.15  30.65 ± 1.18** 22.26 ± 1.95  24.21 ±1.56 % EF 66.84 ± 1.43  43.30 ± 3.38** 64.24 ± 2.52  62.62 ± 2.75^(††) %FS 36.22 ± 1.21  25.84 ± 3.01** 34.70 ± 1.82  34.29 ± 2.09^(††) Datarepresents the Mean ± SEM. *p < 0.05; **p < 0.01 denotes significancecompared with the vehicle treated WT mice. ^(††)p < 0.01 denotessignificance compared with the vehicle treated Ptpn11^(D61G/+) mice. Allp values were derived using 2-way ANOVA (Tukey multiple comparison).IVS, Intraventricular septum wall thickness; LVID, left ventricularinternal dimension; LVPW, left ventricular posterior wall thickness; LVvol, left ventricle volume; EF, ejection fraction; FS, fractionalshortening; d, diatole; s, systole.

TABLE 3 Echocardiography parameters of prenatal vehicle- ordasatinib-treated WT and Ptpn11D^(61G/+) mice at P56. Vehicle DasatinibWT (n = 8) D61G/+ (n = 8) WT (n = 8) D61G/+ (n = 8) IVS,d (mm) 0.71 ±0.03  0.72 ± 0.05  0.69 ± 0.04 0.70 ± 0.03 IVS,s (mm) 1.21 ± 0.10  1.04± 0.04  1.07 ± 0.05 1.02 ± 0.05 LVID,d (mm) 3.83 ± 0.09  3.85 ± 0.11 4.21 ± 0.07* 4.06 ± 0.11 LVID,s (mm) 2.42 ± 0.07  2.92 ± 0.10***  3.04± 0.10*** 3.08 ± 0.07 LVPW,d (mm) 0.80 ± 0.02  0.78 ± 0.04  0.71 ± 0.040.68 ± 0.02 LVPW,s (mm) 1.25 ± 0.04  0.95 ± 0.05***  0.95 ± 0.05*** 0.93± 0.06 LV vol,d (mm³) 67.13 ± 3.87  69.58 ± 5.82 79.32 ± 1.22 73.00 ±3.42  LV vol,s (mm³) 19.76 ± 1.25  31.76 ± 2.73*** 40.34 ± 1.60*** 37.43± 2.08  % EF 70.35 ± 1.23  49.79 ± 2.37*** 50.96 ± 2.38*** 51.83 ± 1.60 % FS 39.98 ± 1.26  26.53 ± 1.30*** 25.84 ± 1.47*** 26.22 ± 1.01  Datarepresents the Mean ± SEM. *p < 0.05; ***p < 0.001 denotes significancecompared with the vehicle treated WT mice. All p values were derivedusing 2-way ANOVA (Tukey multiple comparison). IVS, Intraventricularseptum wall thickness; LVID, left ventricular internal dimension; LVPW,left ventricular posterior wall thickness; LV vol, left ventriclevolume; EF, ejection fraction; FS, fractional shortening; d, diatole; s,systole.

Dasatinib was effective in curtailing NS-Shp2 effects on CHDpost-developmentally. NS mice were treated with dasatinib (0.1mg/kg/day) from P10 until P42 (FIG. 14f ). In addition to thepresentation of CHD, NS humans and mice show growth retardation, facialdismorphism and splenomegaly similar to that of the human disease.Although NS mice were found to exhibit reduced growth, facialdismorphism and splenomegaly, dasatinib treatment did not improve any ofthese NS-related pathologies (FIGS. 19-21). Moreover, no evidence ofliver damage was detected in either wild type or NS mice treated withdasatinib (FIG. 22).

However, upon examination of cardiac parameters, dasatinib-treated NSmice (P42) had completely restored cardiac functionality as determinedby measures of EF and FS (FIGS. 14g and 14h and Tables 4-5). Remarkably,when cardiac function was assessed in NS mice at a later timepoint,after which dasatinib treatment had been discontinued for 2 weeks,similar levels of cardiac functional improvements as compared withvehicle-treated wild type controls was observed (FIGS. 14i and 14j ).Additional cardiac parameters were also assessed by invasivehemodynamics and these results showed that aortic blood pressure andleft ventricular pressure were significantly restored indasatinib-treated NS mice (FIGS. 14k-14n and Table 6).

Taken together, these data demonstrate that dasatinib, when administeredto NS mice post-developmentally at a dose that is sub-therapeutic forits use as a treatment of CML, provides selective efficacy forpreventing cardiac failure in NS mice. Interestingly, the improvement incardiac function does not appear to be transient, since removal ofdasatinib did not reverse the restoration of cardiac function in NSmice.

TABLE 4 Echocardiography parameters of postnatal vehicle- ordasatinib-treated WT and Ptpn11D^(61G/+) mice at P42. Vehicle DasatinibWT (n = 7) D61G/+ (n = 6) WT (n = 6) D61G/+ (n = 6) IVS,d (mm) 0.65 ±0.06  0.54 ± 0.04 0.71 ± 0.05  0.66 ± 0.05 IVS,s (mm) 1.06 ± 0.06  1.02± 0.09 1.13 ± 0.06  1.04 ± 0.07 LVID,d (mm) 3.83 ± 0.17  3.55 ± 0.133.80 ± 0.11  3.63 ± 0.07 LVID,s (mm) 2.36 ± 0.18  2.96 ± 0.07** 2.45 ±0.05  2.50 ± 0.08^(†) LVPW,d (mm) 0.72 ± 0.03  0.77 ± 0.02 0.81 ± 0.04 0.71 ± 0.03 LVPW,s (mm) 1.19 ± 0.07  0.95 ± 0.05* 1.25 ± 0.07  1.09 ±0.06 LV vol,d (mm³) 68.43 ± 6.10  56.10 ± 4.28 65.87 ± 2.78  57.35 ±2.19 LV vol,s (mm³) 20.15 ± 3.73  30.90 ± 1.73** 22.24 ± 0.63  24.67 ±1.18 % EF 65.11 ± 2.42  46.44 ± 5.16** 65.70 ± 0.82  60.93 ± 2.06^(†) %FS 35.04 ± 1.69  24.39 ± 2.92** 35.52 ± 0.69  33.08 ± 1.33^(†) Datarepresents the Mean ± SEM. *p < 0.05; **p < 0.01 denotes significancecompared with the vehicle treated WT mice. ^(†)p < 0.05 denotessignificance compared with the vehicle treated Ptpn11^(D61G/+) mice. Allp values were derived using 2-way ANOVA (Tukey multiple comparison).IVS, Intraventricular septum wall thickness; LVID, left ventricularinternal dimension; LVPW, left ventricular posterior wall thickness; LVvol, left ventricle volume; EF, ejection fraction; FS, fractionalshortening; d, diatole; s, systole.

TABLE 5 Echocardiography parameters of postnatal vehicle- ordasatinib-treated WT and Ptpn11D^(61G/+) mice at P56. Vehicle DasatinibWT (n = 9) D61G/+ (n = 9) WT (n = 6) D61G/+ (n = 6) IVS,d (mm) 0.71 ±0.03  0.69 ± 0.05 0.69 ± 0.03  0.72 ± 0.04 IVS,s (mm) 1.17 ± 0.10  1.01± 0.04 1.19 ± 0.06  1.18 ± 0.05 LVID,d (mm) 3.86 ± 0.08  3.86 ± 0.103.92 ± 0.09  3.93 ± 0.08 LVID,s (mm) 2.43 ± 0.06  2.95 ± 0.10*** 2.52 ±0.10  2.60 ± 0.07^(†) LVPW,d (mm) 0.78 ± 0.02  0.77 ± 0.04 0.80 ± 0.06 0.65 ± 0.03 LVPW,s (mm) 1.22 ± 0.05  0.96 ± 0.04*** 1.18 ± 0.05  1.19 ±0.05^(†) LV vol,d (mm³) 69.38 ± 3.40  69.22 ± 5.22 64.06 ± 2.65  65.60 ±2.44 LV vol,s (mm³) 20.21 ± 1.05  32.80 ± 2.76*** 23.06 ± 2.21  24.85 ±1.68^(†) % EF 68.72 ± 2.09  50.92 ± 2.50*** 65.89 ± 1.74  64.71 ±1.67^(†††) % FS 39.31 ± 1.13  25.64 ± 1.53*** 36.63 ± 1.20  34.92 ±1.23^(†††) Data represents the Mean ± SEM. ***p < 0.001 denotessignificance compared with the vehicle treated WT mice. ^(†)p < 0.05;^(†††)p < 0.001 denotes significance compared with the vehicle treatedPtpn11^(D61G/+) mice. All p values were derived using 2-way ANOVA (Tukeymultiple comparison). IVS, Intraventricular septum wall thickness; LVID,left ventricular internal dimension; LVPW, left ventricular posteriorwall thickness; LV vol, left ventricle volume; EF, ejection fraction;FS, fractional shortening; d, diatole; s, systole.

TABLE 6 Hemodynamic analysis parameters of postnatal vehicle- ordasatinib-treated WT and Ptpn11D^(61G/+) mice at P56. Vehicle DasatinibWT (n = 8) D61G/+ (n = 9) WT (n = 8) D61G/+ (n = 9) Systolic pressure(mmHg) 142.2 ± 9.3  102.8 ± 3.1*** 146.2 ± 5.1  129.3 ± 6.2^(†)Diastolic pressure (mmHg)  89.1 ± 5.0   61.8 ± 2.2***  95.1 ± 4.2   84.0± 3.5^(††) Pulse pressure (mmHg)  67.2 ± 5.3   53.0 ± 1.5  65.2 ± 4.6  63.7 ± 4.4 Mean arterial pressure (mmHg) 106.8 ± 6.3   75.5 ± 2.4***112.1 ± 4.0   96.8 ± 4.2^(††) Left ventricle pressure (mmHg) 137.9 ± 8.0 105.6 ± 3.6** 136.5 ± 2.5  127.9 ± 4.5^(†) End diastolic pressure(mmHg)  7.6 ± 1.8   10.5 ± 0.9  8.7 ± 1.3   11.1 ± 0.7 +dp/dt (mmHg/s) 8497 ± 556  5850 ± 282**  8626 ± 476  7718 ± 498^(†) −dp/dt (mmHg/s)−6075 ± 235 −5354 ± 538 −6529 ± 847 −6189 ± 626 Data represents the Mean± SEM. **p < 0.01; ***p < 0.001 denotes significance compared with thevehicle treated WT mice. ^(†)p < 0.05; ^(††)p < 0.01 denotessignificance compared with the vehicle treated Ptpn11^(D61G/+) mice. Allp values were derived using 2-way ANOVA (Tukey multiple comparison).

To provide further insight into the nature of the cardiac phenotypeexhibited in dasatinib-treated NS mice, gross morphological andhistological examination of these hearts was undertaken. NS mice havelower heart weights as compared with wild type mice (FIG. 15a ). Heartweight to body weight ratios were significantly increased in NS mice(FIG. 15b ). Histological analysis also revealed that NS mice haddilated cardiomyopathy (DCM), shown by significantly reduced leftventricular septum wall thickness and increased left ventricular chamberdimension in systole (FIG. 15c and Table 4).

As expected, histological examination of cardiac tissue revealeddisorganized myofibrillar structures in the left ventricle wall ofvehicle-treated NS mice (FIG. 15d ). In contrast, dasatinib-treated NSmice showed a profound reversal of all of these pathologic cardiacphenotypes to levels that were essentially similar to that ofvehicle-treated wild type mice (FIGS. 15a-15d ).

Another hallmark of the failing heart is the acquisition of cardiacfibrosis. Consistent with the notion that dasatinib treatment conferredprotection against cardiac failure in the NS heart, fibrosis indasatinib-treated NS mouse hearts was markedly reduced as compared withvehicle-treated wild type mice at the histological level consistent withthe reduced mRNA expression levels of fibrotic genes Col1a2 and Col3a1(FIGS. 15d-15f ). The deposition of fibrotic components, such ascollagens, encoded for by Col1a2 and Col3a1 genes, is associated withheart failure. Therefore, the reduction in the expression of Col1a2 andCol3a1 is consistent with the correction of heart failure by low-dosedasatinib treatment.

The re-expression of cardiac structural proteins, such as αMyosin HeavyChain (MYH6) and βMyosin Heavy Chain (MYH7) genes, is indicative ofcardiomyopathy. In particular, inactivation of MYH6 and activation ofMYH7 represent features of cardiac reprogramming that support thedevelopment of cardiomyopathy (Morita et al., J Clin Invest 115 (2005)).MYH6 expression was significantly downregulated in vehicle-treated NSmice as compared with wild type mice. Dasatinib treatment resulted in anequivalent level of MYH6 expression in wild type and NS mice (FIG. 15g). MYH7 was prominently re-expressed in vehicle-treated NS mice and thiswas completely normalized back to vehicle wild-type treated levelsfollowing dasatinib treatment (FIG. 15h ).

The effects of dasatinib to ameliorate cardiac failure in NS mice wasfurther bolstered by the assessment of Atrial naturietic peptide (Anp)and Brain natriuetic peptide (Bnp). Both Anp and Bnp mRNA expressionlevels were significantly upregulated in NS mice as compared withvehicle-treated wild type controls (FIGS. 15i and 15j ). In contrast,dasatinib-treated NS mice were completely rescued from the elevated mRNAexpression levels of both Anp and Bnp (FIGS. 15i and 15j ).Collectively, these results support the conclusion that Src familykinase activity plays an integral role in the development ofNS-associated CHD.

In order to determine whether the effects of dasatinib on NS cardiacfunction were intrinsic to the myocardium, calcium (Ca²⁺) mediated forcedynamics were measured in isolated cardiomyocytes from vehicle- anddasatinib-treated wild type and NS mice. Isolated cardiomyocytes werecharacterized simultaneously for Ca²⁺ handling and contraction kineticsunder electrical pacing. Relative calcium release (R_(mag) Ca²⁺) was 55%higher in NS cardiomyocytes compared with wild type cardiomyocytes andthis difference was substantially ameliorated in dasatinib-treated NSmice (FIGS. 16a and 16b ).

Cardiomyoctyes from vehicle-treated NS mice showed deficits incontractility, with peak shortening that was 22% smaller thanvehicle-treated wild type mice (FIGS. 16a and 16c ). This result isstriking in light of the highly significant increase in Ca²⁺ releaseobserved in these same cardiomyoctyes relative to wild type, andsuggests a reduction in myofilament Ca²⁺ sensitivity in vehicle-treatedNS cells. However, the fractional shortening of sarcomeres in NScardiomyocytes was significantly impaired and was 22% lower than that inwild type cardiomyocytes (FIGS. 16a-16c and Table 7). Importantly, thesedifferences were completely restored in cardiomyocytes isolated fromdasatinib-treated NS mice (FIGS. 16a-16c and Table 7).

The molecular mechanism for the changes in calcium handling andcontractility was investigated by immunoblotting for thesarco(endo)plasmic reticulum Ca²⁺-ATPase 2 (SERCA2A). In the myocardium,SERCA2A is the predominant isoform responsible for calcium delivery tothe contractile machinery. A hallmark of heart failure is reducedSERCA2A expression, which compromises the delivery Ca²⁺ to contractileproteins thus reducing contraction force.

Strikingly, vehicle-treated NS mice hearts showed significantlydecreased SERCA2A protein expression and increased TnI and TnTexpression compared with wild type (FIGS. 16d-16g ). Consistent with therestoration of cardiac function in dasatinib-treated NS mice, cardiactissue isolated from dasatinib-treated NS mice exhibited a completelynormalized level of SERCA2A expression (FIGS. 16d and 16e ).

The ongoing remodeling that occurs during heart failure as a result ofcompensatory mechanisms to preserve contractility results in theupregulation of contractile proteins, Troponin T (TnT) and Troponin I(TnI). Both TnT and TnI were significantly elevated in vehicle-treatedNS mice as compared with vehicle-treated wild type mice (FIGS. 16d, 16fand 16g ). Dasatinib-treated NS mice showed a complete reversal of thefailing heart phenotype since both TnT and TnI expression levelsreturned to those equivalent to vehicle-treated wild type mice (FIGS.16d, 16f and 16g ). Collectively, these findings clearly demonstratethat post-developmental treatment with dasatinib to a NS mouse modelalleviates the contractile dysfunction of the myocardium.

TABLE 7 Ca2+ excitation-contraction coupling parameters ofcardiomyocytes isolated from the heart of postnatal vehicle- ordasatinib-treated WT and Ptpn11D^(61G/+) mice at P56. Vehicle DasatinibWT D61G/+ WT D61G/+ (n = 131 cell, (n = 128 cells, (n = 111 cells, (n =162 cells, n = 3 mice) n = 3 mice) n = 3 mice) n = 3 mice) DiastolicCa²⁺ (Min F

) 1.05 ± 0.01  1.00 ± 0.01***  0.94 ± 0.01***  0.98 ± 0.01 Systolic Ca²⁺(Max F

) 1.21 ± 0.01  1.24 ± 0.01  1.06 ± 0.01***  1.12 ± 0.01^(†††) R_(mag)Ca²⁺ (ΔF

) 0.16 ± 0.01  0.25 ± 0.01***  0.11 ± 0.01***  0.14 ± 0.01^(†††) Ca²⁺TTP (ms, F

) 42.99 ± 0.95   40.41 ± 0.76  42.75 ± 1.21  43.22 ± 0.90 Tau Ca²⁺ (ms)109.18 ± 2.67  123.13 ± 1.88*** 105.85 ± 2.35 116.52 ± 2.36 PeakShortening (%) 5.22 ± 0.19  4.09 ± 0.20***  6.04 ± 0.23*  6.59 ±0.18^(†††) Shortening TTP (ms) 64.45 ± 0.82   67.75 ± 0.97*  71.52 ±0.90***  69.32 ± 0.79 Shortening RT50 (ms) 35.20 ± 0.70   38.74 ± 1.03* 41.11 ± 0.96***  37.02 ± 0.62^(†) Shortening RT90 (ms) 96.47 ± 3.60 112.16 ± 4.35* 113.38 ± 4 18*  88.24 ± 3.23 Data represents the Mean ±SEM. *p < 0.05; ***p < 0.001 denotes significance compared with thevehicle treated WT cardiomyocytes. ^(†)p < 0.05; ^(†††)p < 0.001 denotessignificance compared with the vehicle treated Ptpn11^(D61G/+)cardiomyocytes. All p values were derived using 2-way ANOVA (Tukeymultiple comparison). TTP, Time to peak; RT50, Time from peak tension to50% relaxation; RT90, Time from peak tension to 90% relaxation.

indicates data missing or illegible when filed

Previously, it was determined that the transmembrane glycoprotein PZRwas the most aberrantly hypertyrosyl phosphorylated protein in thehearts of NS mice (Eminaga et al., J Biol Chem 283, 15328-15338 (2008).PZR is a Shp2 binding protein and SFK substrate. PZR hyper tyrosylphosphorylation is a direct result of enhanced NS-mediated Srcsignaling. These data suggest that c-Src functions in the propogation ofNS-related CHD.

The data presented in FIGS. 25a-25f represents dasatinib treatmentstarting at post-natal day 10 (P10) for 6 weeks (P42) in wild type (WT)and NSML (Ptpn11^(Y279C/+)) mice. Ptpn11^(Y279C/+) were obtained fromDr. Kontaridis (Beth Israel Deaconess Hospital, Boston, Mass.) and bredas described (Marin, et al, J Clin. Invest., 121:1026-1043(2011)). Micewere treated with either vehicle or dasatinib at a dose of 0.1 mg/kg/dayfor 6 weeks after which dasatinib treatment was discontinued and micesacrificed two weeks later. At the completion of the study, wild typeand Ptpn11^(Y279C/+) mice were sacrificed and total RNA from the heartswas isolated and qPCR was performed for the detection of mRNA expressionfor genes involved in the development of cardiomyopathy, Myh6 and Myh7and the development of cardiac fibrosis, col1a2 and Col3a1.

As shown, vehicle-treated Ptpn11^(Y279C/+) mice, by 6 weeks of age,already began to show signs of tissue cardiomyopathy as evidenced byincreased expression of ANP, Myh6 and Myh7 (FIGS. 25d and 25e ). Inaddition, there was an accompanying increase in cardiac fibrosis inPtpn11^(Y279C/+) mice as compared with vehicle-treated-wild type mice(FIGS. 25a and 25b ). However, dasatinib-treated Ptpn11^(Y279C/+) miceshowed complete restoration back to wild type levels of expression ofANP, Myh6 and Myh7. Significantly, the ratio of Myh6/Myh7, whichrepresents the switch in fetal/adult myosin contractile genes, was alsorestored to wild type levels in dasatinib-treated Ptpn11^(Y279C/+) mice(FIG. 25f ). Together, these data demonstrate the effectiveness of lowdose dasatinib treatment in correcting Noonan syndrome with multiplelentigines (NSML)-related cardiomyopathy at the molecular level.

This is the first evidence for the involvement of c-Src signaling in thepathogenesis of NS. Other groups have reported that Shp2 lies upstreamof the SFKs in a phosphatase-dependent manner (Zhang et al., Mol Cell13, 341-355 (2004)). Here a distinct mechanism that invokes the “open”conformation of NS-Shp2 mutants providing enhanced PTP-Shp2/Src-SH3binding and localization to promote c-Src signaling is demonstrated.Post-development administration of dasatinib at sub-therapeutic dosesrequired for the treatment of CML was sufficient to restore cardiaccontractility and function. These data strongly implicate c-Src as acentral mediator of NS-mediated pathogenesis. At the low doses ofdasatinib, the Src pathway appeared to be selectively affected, whilstERK1/2 signaling, at least in the myocardium, was negligibly inhibited.However, it is conceivable that ERK1/2 exists in a sub-set of specificcells in the heart that are affected by dasatinib treatment.

Importantly, analysis of calcium-mediated contraction coupling ofcardiomyocytes isolated from dasatinib-treated NS mice clearlydemonstrated that these cells were a site of action through whichdasatinib inhibition of c-Src exerted its effect on the contractilemachinery. Consistent with previous observations NS-Shp2 mutantsincreased Ca²⁺ signaling in the myocardium (Uhlen, et al. PNAS 103,2160-2165 (2006)). Interestingly, myocardium derived from NS miceexhibited reduced contractility, suggesting a reduced sensitivity at thelevel of Ca²⁺-mediated force contraction which can be explained, atleast in part, by the reduced levels of SERCA2A expression.

In summary, a novel and unanticipated therapeutic strategy for thetreatment of PTPN11-mediated CHD is described herein. These dataidentify the Src family of kinases as a class of targets that mediatePTPN11-related CHD. A therapeutic strategy of “low dose” dasatinib, orother c-Src inhibitors, may open up new avenues for the treatment ofheart disease.

Other Embodiments

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1. A method of treating a cardiovascular disease or condition havingaberrant protein tyrosine phosphorylation in a subject, comprisingadministering a low-dosage of a tyrosine kinase inhibitor to a subjectin need thereof, wherein the tyrosine kinase inhibitor decreasesaberrant levels of tyrosine phosphorylation and improves at least onecardiac function in the subject.
 2. A method of treating congenitalheart disease comprising administering a low-dosage of a tyrosine kinaseinhibitor to a subject in need thereof, wherein the tyrosine kinaseinhibitor decreases aberrant levels of tyrosine phosphorylation andimproves at least one cardiac function in the subject.
 3. A method oftreating a cardiovascular disease or condition associated with aRASopathy having aberrant protein tyrosine phosphorylation comprisingadministering a low-dosage of a tyrosine kinase inhibitor to a subjectin need thereof, wherein the tyrosine kinase inhibitor decreasesaberrant levels of tyrosine phosphorylation and improves at least onecardiac function in the subject.
 4. The method of claim 2, wherein thecongenital heart disease is associated with a RASopathy.
 5. The methodof claim 3, wherein the cardiovascular disease or condition iscongenital heart disease.
 6. The method of claim 3, wherein theRASopathy is selected from the group consisting of NeurofibromatosisType 1, Noonan syndrome, Noonan syndrome with multiple lentigines(Leopard syndrome), capillary malformation-arteriovenous malformationsyndrome, Costello syndrome, cardio-facio-cutaneous syndrome, and Legiussyndrome.
 7. The method of claim 1, wherein the low-dosage is in therange of about 175 fold to about 250 fold lower than a chemotherapeuticdosage of the tyrosine kinase inhibitor.
 8. The method of claim 1,wherein the cardiac function is selected from the group consisting ofmyofibrilar organization, cardiomyocyte contractility, SERCA2Aexpression, and cardiac fibrosis.
 9. The method of claim 1, wherein thetyrosine kinase inhibitor is selected from the group consisting ofafatinib, axitinib, bosutinib, cabozantinib, cediranib, ceritinib,crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, gefitinib,ibrutinib, imatinib, lapatinib, lenvatinib, lestaurtinib, nilotinib,nintedanib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib,semananib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib,trametinib, vandetanib, and vemurafenib.
 10. The method of claim 1,wherein the tyrosine kinase inhibitor is a Src family tyrosine kinaseinhibitor.
 11. The method of claim 10, wherein the Src family tyrosinekinase inhibitor is selected from the group consisting A419259, AP23451,AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675,CU201, ENMD 2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765,PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656,TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228,altenusin, bosutinib, damnacanthal, dasatinib, herbimycin A, indirubin,neratinib, lavendustin A, pelitinib, piceatannol, saracatinib, SrcI1,and analogs thereof.
 12. The method of claim 1, wherein the subject is apediatric patient.
 13. The method of claim 12, wherein the pediatricsubject is less than 12 years of age.
 14. The method of claim 1, whereinthe subject is greater than 18 years of age.
 15. The method of claim 1,wherein the aberrant levels of tyrosine phosphorylation compriseaberrant levels of tyrosine phosphorylated Protein Zero-Related (PZR).16. The method of claim 15, wherein the low-dosage tyrosine kinaseinhibitor decreases PZR tyrosine phosphorylation.
 17. The method ofclaim 1, wherein the low-dosage tyrosine kinase inhibitor provides ananti-fibrotic effect in cardiac tissue to the subject.
 18. A compositioncomprising a low-dosage tyrosine kinase inhibitor, wherein thelow-dosage tyrosine kinase inhibitor is capable of decreasing tyrosinephosphorylation and improving at least one cardiac function in a subjectin need thereof.
 19. The composition of claim 18, wherein the low-dosagetyrosine kinase inhibitor decreases aberrant tyrosine phosphorylation ofa transmembrane glycoprotein.
 20. The composition of claim 19, whereinthe transmemberane glycoprotein is Protein Zero-Related (PZR).
 21. Thecomposition of claim 18, wherein the low-dosage tyrosine kinaseinhibitor is in the range of about 175 fold to about 250 fold lower thana chemotherapeutic dosage of the tyrosine kinase inhibitor.
 22. Thecomposition of claim 18, wherein the cardiac function is selected fromthe group consisting of myofibrilar organization, cardiomyocytecontractility, SERCA2A expression, and cardiac fibrosis.
 23. Thecomposition of claim 18, wherein the low-dosage tyrosine kinaseinhibitor is selected from the group consisting of afatinib, axitinib,bosutinib, cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib,dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, imatinib,lapatinib, lenvatinib, lestaurtinib, nilotinib, nintedanib, palbociclib,pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus,sorafenib, sunitinib, temsirolimus, tofacitinib, trametinib, vandetanib,and vemurafenib.
 24. The composition of claim 18, wherein the tyrosinekinase inhibitor is a Src family tyrosine kinase inhibitor.
 25. Thecomposition of claim 24, wherein the Src family tyrosine kinaseinhibitor is selected from the group consisting A419259, AP23451,AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675,CU201, ENMD 2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765,PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656,TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228,altenusin, bosutinib, damnacanthal, dasatinib, herbimycin A, indirubin,neratinib, lavendustin A, pelitinib, piceatannol, saracatinib, SrcI1,and analogs thereof.
 26. The composition of claim 18, wherein thelow-dosage tyrosine kinase inhibitor provides an anti-fibrotic effect incardiac tissue.
 27. A pharmaceutical composition comprising thecomposition of claim 18 and a pharmaceutically acceptable carrier. 28.Use of the composition of claim 18 in the manufacture of a medicamentfor the treatment of cardiovascular disease or condition in a subject.